1

STREPTOCOCCI

SYSTEMATICS

Domain : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Lactobacillales
Family : Streptococcaceae
Genus : Streptococcus
Species : Str. agalactiae, Str.dysgalactiae, Str. equi subsp.
zooepidemicus, Str.uberis, Str. equi subsp equi, Str.suis, Str.
canis, Str. pyogenes (human)
HISTORY

Rivolta (1873) described chain forming organisms in pus from a case of strangles in horses.
In 1878-79, Pasteur recognized this organism as a pus-forming agent. In 1903, Hugo Schottmuller
introduced blood to differentiate various types of hemolysis. In 1928, Rebecca Lancefield reported a
serological method of grouping streptococci.

HABITAT

Streptococci are worldwide in distribution. Most of the streptococci of Veterinary interest live
as commensals in the mucosa of the upper respiratory and lower urogenital tracts. They do not
survive for long away from the animal hosts.

MORPHOLOGY

Streptococci are gram positive, spherical or ovoid cells, arranged in chains or pairs. Chain
formation is due to the cocci dividing in one plane only and the daughter cells failing to separate
completely. Each coccus is about 1 m in diamter. They are facultative anaerobes, catalase negative,
oxidase-negative, and non-spore forming and non-motile with the exception of the some of the
enterococci.

Capsulation is not a regular feature of streptococci but some strains of Str.pyogenes and
some group C strains have capsules composed of hyaluronic acid while polysaccharide capsules are
encountered in members of group B and D. Protoplasts (L-forms) may be induced by pencillin or
phage associated lysine and may be propagated on hypertonic media.

CLASSIFICATION OF STERPTOCOCCI

I. Several systems of classification have been employed. Based on growth characteristics, type of
haemolysis and biochemical activities they can be divided into 6 principal categories.

1. Pyogenic Streptococci : Str. pneumoniae, Str.pyogenes, Str.equi, Str.dysgalactiae

2. Oral Streptococci : Str.salivarius
3. Enterococci : Str.faecalis, Str.avium, Str. gallinarum
4. Lactic acid Streptococci : Str.lactis
5. Anaerobic Streptococci : Str. monbillorum
6. Other Streptococci : Str.bovis, Str.uberis, Str.equi subsp zooepidemicus

II. The aerobic and facultative anaerobic Streptococci are classified, based on their haemolytic
properties. Brown (1919) established this method by employing meat infusion peptone agar with 5%
horse blood. He recognized three types of reactions.
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Alpha - haemolytic Streptococci

They produce a greenish discolouration with partial haemolysis around the colonies. The
zone of lysis is small (1or2 mm wide), within which the unlysed erythrocytes are seen. These ά -
Streptococci are generally commensals in the throat. Because of the distinctive green color, they
produce; they are called as greening or viridans Streptococci.

Eg. Str.pneumoniae

Beta haemolytic Streptococci

Beta haemolytic Streptococci produce a sharply defined, clear, colourless zone of haemolysis,
2 or 4mm wide, within which the red cells are completely lysed. Most of the pathogenic Streptococci
fall into the beta group and are called as the haemolytic Streptococci.

Gamma or non-haemolytic Streptococci

Gamma or non-haemolytic Streptococci produces no change in the medium. The gamma

Streptococci includes the faecal Streptococci (Str. faecalis) and related species. They are called the
enterococcus or indifferent Streptococci.

Haemolytic streptococci of group A are known as Str.pyogenes. These may be further

subdivided into types based on the protein (M, T and R) antigens present on the cell wall. The M
protein is acid and heat labile and the T protein is acid labile and trypsin resistant. Some of the
lancefield groups may be further subdivided by means of the agglutination test and designated by
Arabic numbers-Griffth typing.

III. Another important way in which the beta haemolytic Streptococci were classified by Rebecca
lancefield (1933) based on the nature of a carbohydrate (C) antigen on the cell wall. They are known
as Lancefield groups, 19 of which have been identified so far and named by the capital letters A-U
(without I and J)

Lancefield group Species Host Disease

Streptococci Humans Scarlet fever, Septic sore throat,

A pyogenes erysipelas, abscesses and
rheumatic fever

S. agalactiae Cattle, sheep& Chronic mastitis

goats
B
Human & dogs Neonatal septicaemia

Cats Kidney and uterine infections

S. dysgalactiae Cattle Acute mastitis

C
Lambs Polyarthritis

S. dysgalactiae Horse Abscesses, endometritis and

subsp. Equismilis mastitis

S. equi subsp. Horse Strangles, genital and

Equi suppurative conditions,
Mastitis and purpura
Haemorrhagica
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S. equi subsp. Horse Mastitis, abortion, secondary

Zooepidemicus Pneumonia and navel
infections

Cattle Metritis & Mastitis

Pigs Septicaemia & arthritis in 1-3

wk old piglets

Poultry Septicaemia & Vegetative

Endocarditis

Lambs Pericarditis and Pneumonia

Enterococcus Many species Opportunistic infections
D.
faecalis

S. equines and Many species Opportunistic infections

Str. Bovis

E. Str. Porcinus Pigs Jowl abscesses &

lymphadenitis
(P,U,V)

Str. Canis Carnivores Neonatal septicaemia,

genital, skin and wound
G
infections

Cattle Occasional mastitis

Lactococcuc Cattle unknown
N
lactis

Enterococcus Many species unknown

Q
avium

S.suis type 2 Pigs Meningitis and arthiritis

R
(4 to6months)

S.suis type 1 Pigs (2 to 4 wks Meningitis and arthritis

S
old)

Ungroupable S.uberis Cattle Mastitis

S.pneumoniae Guinea pigs, rats Pneumonia

and primates

. Haemolytic streptococci of group A are known as Str.pyogenes. These may be further

subdivided into types based on the protein (M, T and R) antigens present on the cell wall. The M
protein is acid and heat labile and the T protein is acid labile and trypsin resistant. Some of the
lancefield groups may be further subdivided by means of the agglutination test and designated by
Arabic numbers-Griffth typing.

CULTURAL CHARACTERISTICS

It is an aerobe and facultative anaerobe, growing best at a temperature of 37°C. They grow
best in media enriched with blood, serum and fermentable carbohydrates. On blood agar after
incubation for 24hrs small, circular, semitransparent colonies with an area of clear haemolysis are
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produced. Virulent strains from fresh isolates produce ‘matt’ (finely granular) colony and avirulent
strains form ‘glossy’ colonies. Strains producing capsules form mucoid colonies.

In glucose or serum broth, growth occurs as a granular turbidity with a powdery deposit. No
pellicle is formed.

In Edward’s medium (selective media) it produces dewdrop like black colonies. Edward’s
medium containing blood agar, crystal violet and aesculin (differentiate among different species of
streptococci which do or do not hydrolyse aesculin). Str. pneumonia is alpha haemolytic, produces
mucoid or flat colonies with smooth borders and a central concavity after 48-72hrs on blood agar
(draughts man colonies). Ability to grow in 0.1 % tellurite broth is characteristic of Str. faecalis.
Majority of streptococcal species do not grow on MacConkey agar except Enterococcus faecalis.

BIOCHEMICAL PROPERTIES

Streptococci are catalase and oxidase negative. Ferment several sugars producing acid but
no gas. They ferment sorbitol, trehalose, lactose, maltose, dextrin, and mannitol. Gelatin not liquefied.
Nitrates not reduced, Indole is negative. They are not soluble in 10% bile unlike pneumococci.

RESISTANCE

It is a delicate organisms easily destroyed by heat (54°C for 30 minutes). It can survive in
dust for several weeks, if protected from sunlight. It is rapidly inactivated by antiseptics. It is more
resistant to crystal violet and susceptible to sulphonamides and other antibiotics. Sensitivity to
bacitracin is employed as a convenient method for differentiating Str. pyogenes from other hemolytic
streptococci.

ANTIGENICITY

The hyaluronic acid capsule of Str. pyogenes inhibits phagocyotosis. The cell wall is
composed of an outer layer of fimbria containing proteins and lipoteichoic acid, a middle layer of
group specific carbohydrate and an inner layer of peptidoglycon. The Cell wall polysaccharide has
been shown to have a toxic effect on connective tissue in experimental animals.

The peptidoglycan is responsible for cell wall rigidity. Several protein antigens have been
identified in the cell wall (M, T and R). They are responsible for type specificity in Str.pyogenes.
Among these the M protein is act as a virulence factor by inhibiting phagocytosis.

Hair-like pili project through the capsule of group A streptococci. The pili consist partly of M
protein and are covered with lipoteichoic acid, which is important in the attachment of streptococci to
epithelial cells.

TOXINS AND OTHER VIRULENCE FACTORS

Streptococci form several exotoxins and enzymes, which contribute to its virulence

Extra cellular toxins

a) Haemolysins

Streptolysins O and S are produced by groups A, C and G.

1) Streptolysin O

It is so called because it is oxygen labile. It is an antigenic protein and is active in the reduced
form. On blood agar, its activity is seen only in pour plates and not in surface cultures. It is also heat
 5

labile. It is lethal on I/V inj. into animals and has a specific cardiotoxic activity. It is a general cytotoxin.
Red cells of all animal species except mouse are lysed. Streptolysin O is antigenic and
antistreptolysin regularly appears in sera following Streptococcal infection. Estimation of this
antibody (ASO) titre is a standard serological procedure for the retrospective diagnosis of infection
with Str.pyogenes.

2) Streptolysin S

It is an oxygen stable haemolysin and so is responsible for the beta haemolysis seen around
streptococcal colonies on the surface of blood agar plates. It is called Streptolysin S since it is soluble
in serum. Addition of serum to broth increased the yield of haemolysin. It is protein but not antigenic.
It has been shown experimentally to be nephrotoxic.

b) Erythrogenic toxin (Streptococcal pyogenic exotoxins/ Dick toxin)

Four erythrogenic toxins are known and most strains of Str. pyogenes produce one or more.
They are pyogenic and enhance susceptibility to lethal shock by endotoxin. The toxin is thermostable
and antigenic. The intradermal injection in rats leads to development of erythema. This reaction is
called as “Dick test” or Schultz-charlton reaction and it is useful for diagnosis of scarlet fever.

c) Enzymes

i) Streptokinase: (Fibrinolysin)

Filtrate of Streptococci gp, A, E & G produces fibrinolysin. This toxin promotes the lysis of
fibrin clots by activating a plasminogen. Fibrinolysin plays a biological role in streptococcal infections
by breaking down the fibrin barrier around the lesions and facilitating the spread of infection.

ii) Deoxyribonucleases: (Streptodornase)

This causes depolymerisation of DNA, pyogenic exudates contains large amount of DNA,
derived from the nuclei of necrotic cells. Streptodornase helps to liquefy the thick pus and may be
responsible for the thin serous character of streptococcal exudates. Four antigenically distinct
streptodornase (A, B, C & D) have been recognized.

iii) Hyaluronidase:

This enzyme breaks down the hyaluronic acid of the tissues. This might favour the spread of
infection along intercellular spaces. Streptococci possess a hyaluronic acid capsule and also
elaborate a hyaluronidase- a seemingly self-destructive process. This is produced by group A, C, G
and B Streptococci.

iv) Proteinase:

This is another instance of an apparently self-destructive enzyme, since it is capable of

breaking down the M protein, streptokinase and hyaluroindase. The enzyme is, however, produced
only under special conditions such as an acid pH (5.5 –6.5). Such conditions may be produced by
tissue destruction, as in abscesses. Most strains of Str. pyogenes forms proteinase.

v) Neuraminidase:

This activity is detected in streptococci groups A, B, C, G and L. This enzyme is a virulence

factor for pathogens surviving on mucosal surface. In addition to this M types of Str.pyogenes
produce NADse and other many strains also produce esterase, amylase and N-acetyl
glucosaminidase.
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vi) Serum opacity factor:

When gp. A. Streptococci grown in horse serum it produces an opalescence of the serum.
This opacity factor is a protein. This is a lipoproteinase and opalescence is a result of an
agglomeration of the bacterial antigen.

PATHOGENESIS

The natural habitat of the species of streptococci are skin, nose, throat, digestive and
urogenital tract of man and animals. Str.pyogenes is present in human nose and throat without
causing any disease, while Str.agalactiae and Str.uberis can exist in bovine udder causing mastitis.

Streptococcus  through teat end  Adherence (M protein) & multiplication of

the organism on the epithelium of teat &
duct sinuses

PMN attracted Death of PMN

Involution of fibrin plug
Chronic Secretary Tissue formation in the inflammation and
Mastitis & smaller milk ducks fibrosis
Loss of milk
Producing capacity

PATHOGENICITY

Cattle:

Bovine mastitis: It is caused by Str. agalactiae (group B), Str. dysgalactiae (group C), Str. equi
subsp. zooepidemicus (group. C), Str. uberis (group C, D, E, P, V). Mastitis arises from the
multiplication of streptococci in the teat sinus and extends into the ducts.

It causes parenchymatous mastitis, which is characterized by progressively chronic condition

resulting in fibrosis. In acute stages milk is composed of purulent exudate, dead tissue cells,
coagulated milk protein and bacteria.

Peptostreptococcus indolicus is an anaerobic streptococcus, which is responsible for

summer mastitis in cattle in association with Arcaobacterium pyogenes.

Horse:

Str. equi and str. equisimilis is the main cause of strangles in young horses. It is
characterized by a catarrhal discharge, with inflamation of the nasal mucous membranes, followed by
swelling of pharyngeal LN’s in which abscesses develop. The infection spreads through lymph
channels. It also causes metritis and cervicitis in horses.

Purpura haemorrhagica, considered to be an immune mediated disease, occur in horses 1to 3

weeks after illness.

Bastard strangles – in which abscess developed in many organs. It is a very serious

complication.
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Chicken:

Str. gallinarum causes typical acute septicemia with peritonitis in chicken.

Dogs:

Str. canis is considered to be the cause of acid milk in puppies and canine tonsillitis. It is also
associated with neonatal septicaemia and toxic shock syndrome.

Pigs:

Str. suis causes porcine cervical lymphadenitis and also isolated from pneumonia, septicemia,
arthritis, endocarditis, meningitis and reproductive tract infections. It also causes erosive arthritis in
young pigs.

DIAGNOSIS

It involves clinical, microscopical and bacteriological examination. Palpation of the udder and
supramammary lymphnodes will be helpful in distingusing the chronic and insidious form of mastitis
produced by Str.agalactiae and Str.uberis.

In contrast Str.dysgalactiae and Str.zooepidemicus causes sudden onset of acute

inflammation of one quarter only with an acute systemic disturbance followed by joint infections and
lameness.

Microscopical examination:

When long chains of organism are detected in milk samples from chronic mastitis, it is caused by
Str.agalactiae.

Bacteriological examination:

When 0.1 ml of secretions inoculated on Edward’s medium (blood agar, crystal violet and
aesculin) Str.agalactiae produces blusih-grey colonies and Str.uberis produces dark color colonies.

CAMP test (Christie, Atkins, Munch and Peterson, 1944):

This test is based on the observation that ruminant red blood cells lysed by the beta toxin of
o
staphylococci at 37 C are completely lysed in the presence of Str.agalactiae (group B).

Bile solubility test:

Differentiation between the pneumococcus and Str.viridans organisms can be achieved by

bile solubility and the optochin test. Autolysis of pneumococcal cultures takes place within 15minutes
0
at 37 C in the presence of 10 per cent sodium deoxycholate. These substances have no effect on Str.
viridans organisms.

The optochin test:

The majority of pneumococcal strains are sensitive to optochin (Ethyl hydrocuprein

hydrochloride), whereas Str.viridans organisms are not. This test consist of placing a small circular
piece of filter paper, impregnated with 1:4000 aqueous solution of optochin, in the center of a blood
agar plate after inoculating the test cultures in streaks across the full width of the medium. The
growth of pneumococcal strains will be inhibited to a distance of some 5mm from the circumference
of the filter paper

CONTROL AND PREVENTION

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To treat the Streptococcal infections in animals, including mastitis, the most satisfactory method
is antibiotic therapy using pencillin preparations to other substances because streptococci develop
resistance to pencillin comparatively infrequently.

Vaccines are of very limited value for the immunization of animals against streptococcal
infections, with the possible exception of equine strangles.

STAPHYLOCOCCI

First discovered by Scottish surgeon sir Alexander ogston (1880) in infected tissues. He
named it as staphylococcus” (Greek staphyle, bunch of grapes; KOKKAS, berry).

SYSTEMATICS
Domain : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Bacillales
Family : Staphylococcaceae
Genus : Staphylococcus
Species : Staph. aureus
Staph. intermedius
Staph. hyicus

HABITAT AND ECOLOGY

Staphylococcus occurs worldwide in mammals although the spread of staphylococcal strains

between different animal species is limited. They colonize the nasal cavity, skin and mucous
membranes and can be transient in the intestinal tract. It is an opportunist type organism.

MORPHOLOGY

Spherical cells, 0.8 to 1.0, micrometer in diameter on agar media the cocci are arranged in
grapes like clusters. In broth they occur as small groups, pairs or short chains of not more than four
numbers. They are gram positive, non-motile, non-acid fast and non-sporing and have no flagella.

CULTURAL CHARACTERISTICS

- They are aerobic and facultatively anaerobic.

- They grow at an optimum temperature of 35-37°C.
- Staphylococcus grows in the presence of 7-10% Sodium chloride. Hence a media-
containing salt is selective for this organism (eg; Mannitol Salt agar) and it is highly
inhibitory to many other bacteria particularly gram-negative bacteria.
- In Nutrient agar plate the colonies are round, smooth, glistening, opaque, low, convex,
 9

edge, entire end of a golden yellow or white colour. In nutrient broth a uniform turbidity is
present with powdery sediment.
- Phenolpthaline diphosphate or tellurite agar selectively inhibits non-pathogenic strain.
- Haemolysis on blood agar. Capable of liberating ‘V’ factor into the medium, which favours
the growth of Haemophilus organism.
- Purple agar, containing bromocresol purple as a pH indicator and 1% maltose, is used to
differentiate Sta. aureus and Sta. intermedius.
- Most strains form pigments, hence previously classified as per the colour of the pigment
produced.
Eg: Staph.aureus : yellowish or golden orange pigment.
Staph.albus : white colonies.
Staph.citreus : lemon yellow colour pigment.
- Later on it was found that pigment formation is variable. Hence such classification was
no longer followed.
- In sheep or rabbit blood agar plate “double hemolysis” around colonies are formed with
incubation at 37°C it produces an incomplete haemolysis, which develops into a complete
haemolysis when held at 4°C. This is called as hot cold lysis phenomenon.

RESISTANCE

Staphylococci are the most resistant of the cocci. Usually a temperature of 60°C for half an
hour destroys all the cells. Death is accomplished in 1% phenol in 35 minutes or 10% formaldehyde in
10 minutes.

BIO-CHEMICAL PROPERTIES

Staphylococci aureus produces acid from glucose maltose, mannitol, lactose, and sucrose
and not from salicin, raffinose & inulin. The organism is indole negative positive for NH3, methyl red
and voges – proskauer and catalase. Hydrolyses gelatin and coagulate serum. Negative for oxidase
and H2S production.

ANTIGENIC STRUCTURE

It consists of;

1) Group antigen

a) Carbohydrates
The cell wall of Staph. aureus contains ribitol teichoic acid and Staph. intermedius
contains glycerol teichoic acid.

b) Protein A. is present only in Staph. aureus

2) Type antigen
Proteins other than protein A

Virulence Factors

Three groups of virulence factors

1. Surface components

It consists of capsule, Teichoic acid, which have antiphagocytic ability.

2. Exotoxins

Hemolysin:

Four antigenically distinct hemolysin causes hemolysis of erythrocytes. They are Alpha, Beta,
delta and Epsiton toxin, which produce partial hemolysis (hot – cold hemolysis). The alpha toxin is the
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major toxin in gangrenous mastitis. It causes spasm of smooth muscle and is necrotizing and
potentially lethal.

Leukotoxin:

It has the leukocidal activity and includes  and  toxins. By along so, the organisms may
spread more easily to other parts where they develop secondary lesions.

Enterotoxin:

It is seldom produced by animal strains. There are several antigenically distinct types of heat
stabe enterotoxins. They are not destroyed at 100°C for 80 minutes. They are responsible for food
poisoning in man.

Exfoliative or Dermonecrotoxin:

This toxin causes necrosis of skin by exfoliation and intraepidermal seperation.

Toxic shock syndrome toxin (TSST):

Induce excessive lymphokine production results in tissue damage. Bovine and human strains
of Sta. aureus produce TSST

3. Extracellular enzymes

Coagulase:

On their ability to coagulate plasma they are classified as coagulase positive staph. (CPS) and
coagulase negative staph. CPS is considered to be significant pathogens. It is resistant to heat. The
coagulation of plasma produces a fibrin film on the surface of the organisms, which allows
multiplying.

Hyaluronidase:

It hydrolyses hyaluronic acid, the mucoid ground substance of connective tissue.

Nucleases:

Deoxyribonucleases (DNAse) hydrolyze DNA and Ribonucleases hydrolyze RNA.

Fibrinolysin:

Fibrinolysin is commonly referred to as staphylokinase, is an activator of the plasma system

leading to the breakdown of fibrin.

Lipases and esterases:

They hydrolyze lipids.

Lysozyme:

Hydrolyses the peptidoglycan in the cell wall of many bacteria.

PATHOGENESIS
Capsules
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Normal inhabitant  Tissue damage antiphagocytoic

Protein A activity

Establishment
of staphylococci Protect bacteria Tissue damage
Coagulase Extracellular
Enzymes

PATHOGENICITY

Horse:

Botryomycosis - Infrequent chronic granulomatous lesions involving the udder of the mare,
cow and sow and the spermatic cord of horses.

Cattle:
Mastitis - Staphylococcal bovine mastitis may be chronic, acute and peracute. Gangrenous
mastitis due to  toxin is seen in postparturient cows.

Sheep:

Tick pyemia in lambs occurs in 2-5 week old lambs, which is heavily infected with Ixodes
ricinus. Periorbital eczema is an infection due to abarasion. Staphylococcal dermatitis is due to
scratches from vegetation.

Poultry (Bumbel foot):

A pyogranulomatous process of subcutaneous tissue of foot that involves the joints.

Staphylococcal arthritis and septicemia in turkeys, omphalitis – yolk sac infection, wing rot or
gangrenous dermatitis infection in poultry

Pig:

Exudative epidermitis (greasy pig disease) is an acute generalized infection of suckling and
weaned pigs caused by Sta. hyicus. This disease is characterized by excess sebaceous secretion,
exfoleation and exudation.

Dogs and Cats:

Pyoderma is one of the most common skin diseases of dogs. In addition to this, Otittis
externa and other supuurative conditions are caused by Sta. intermedius. Staphylococcal antigens
produce intense inflammatory reaction and promote persistence of the bacteria.

Other Staphylococcal organisms

S. aureus sub sp. anaerobius causes caseous lymphadenitis. They are anaerobic and catalse
negative.

S. caprae  Goat’s milk

S. gallinarum and S. arlettae  Skin of chickens

S. lentus  Skin of sheep and goats
S. equorum  Skin of horses
S. simulans and S. felis  Cats
S. delphini  Skin of clophins
S. aureus  Staphylococcal scalded skin syndrome
(SSSS) and Toxic shock syndrome (TSS) in
humans
 12

MRSA  Methicillin resistant staphyloccol aureus

DIAGNOSIS

- Direct microscopic examination

- Cultural characters in selective media.
- In case of food poisoning identification of the enterotoxin is diagnostic use
- CPS produces typical reaction in the Hotis test of milk samples. These organisms
produced green or greenish brown colonies with white centres after the milk samples
are incubated for 16-20 hour.
- DNA test and protein A tests are also employed.

PATHOGENICITY TEST

Coagulase test:

When culture added to Rabbit plasma, fibrinogen is converted to fibrin by coagulase enzymes.
In this test, a suspension of staphylococci is mixed with rabbit plasma either on a slide or in a small
tube.

The slide test detects the presence of a bound coagulase or clumping factor on the bacterial
surface. A positive reaction is indicated by clumping of bacteria within 1 to2 min.

The tube test detects the free coagulase or staphylocoagulase, which is secreted by bacteria
into the plasma. It is the definitive test for coagulase production and positive reaction is indicated by
0
clot formation in the tube following incubation at 37 C for 24 hrs.

Phage typing

Phage typing is carried out for epidemiological purposes, particularly for Staph.
aureus strains of bovine mastitis.

Treatment

Penicillin is the drug of choice if strains are susceptible. New synthetic penicillins such as
methicillin, oxacillins are effective. Tetracyclines, bacitracin, nitrofurans, Trimethoprins,
Cephalosporins, enrofloxacins are effective.

Main differentiating characteristics of the gram-positive cocci

Organisms Coagulase Catalase Oxidase

Pathogenic staph. + + -
Non.Pathogenic Staph. - + -
Enterococci - - -
Streptococci - - -
Micrococci - - +
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BACILLUS

B.anthracis causes anthrax in animals and Wool sorter’s disease, hide porter’s disease,
Malignant Pustule in humans. B.cerus causes food poisoning in humans. Other bacillus in this group
is non-pathogenic and they are called as anthracoids. B.licheniformis is an emerging pathogen and it
is implicated in sporadic abortions in cattle and sheep.

SYSTEMATICS

Domain : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Bacillales
Family : Bacilliaceae
Genus : Bacillus
Species : B.anthracis, B.cereus, B.subtilis, B.mycoides,
B.megaterium, B.mesentricus

FAMILY CHARACTERS

They are gram +ve large rods, aerobic (facultative anaerobic), endospore forming, capsulated,
mostly catalase positive and fermentative organisms. They are motile by peritrichous flagella.

BACILLUS ANTHRACIS

HISTORY

- Discovery of the anthrax bacillus is credited to Davaine and Rayer (1863 –1868).
- Considerable historic interest attached to anthrax bacilli;
- Pollender 1849 – Anthrax bacillus was the first pathogenic bacterium observed under the
Microscope
- Davaine 1850 – first communicable disease shown to be transmitted by inoculation of
infected blood
- Koch 1863 – first bacillus to be isolated in pure culture
- Pasteur 1881 – used for the preparation of attenuated vaccine.

MORPHOLOGY

The anthrax bacillus is one of the largest pathogenic bacteria. They are gram+ve, straight, rod
shaped, non-motile organism measuring 4-8 um x 1-1.5um. In cultures the bacilli are arranged end to
end in long chains. The ends of the bacilli are truncated or often concave and somewhat swollen. So
that chain of bacilli presents a bamboo stick appearance (also called as Box - car bacillus - look like
linked rail carriages).

In tissues or in blood smear, it is found singly, in pairs or in short chains, the entire bacilli being
surrounded by capsule. The capsule is polypeptide in nature, being composed of a polymer of d-
glutamic acid. Capsules are not formed under ordinary conditions of culture, but only if the media
contains serum, albumen, charcoal, starch or bicarbonates with reduced partial pressure of carbon
dioxide.

When blood films containing bacilli are stained with polychrome methylene blue for a few
 14

seconds and examined under the microscope, an amorphous purplish material is noticed around the
bacilli. This represents the capsular material and is characteristic of the anthrax bacilli. This is called
as McFadeyan’s reaction. This reaction depends on the degree of heat employed for fixation of a
blood film.

Sporulation occurs readily outside the body in the presence of oxygen. Spores are formed in
culture or in the soil, but never in the animal body during life. Sporulation occurs under unfavourable
conditions for growth and is encouraged by distilled water, 2%NaCl or growth in oxalated agar.
0
Sporulation takes place at an opt.temp.of 25-30 C and in atmosphere containing low partial pressure
of oxygen.

Spores are central, elliptical or oval in shape and are of the same width as the bacillary body.
So that they do not causes bulging of the vegetative cell. The spores do not stain by ordinary methods.
But can be stained with Sudan black B.

CULTURAL CHARACTERS

0
 Grows readily under aerobic and a facultative anaerobic at an opt.temp. of 35-37 C
 On agar plates, irregular, round, raised, dull, opaque, grayish white, 2-3mm in d.m. frosted
glass appearance colonies are produced. Under the low power microscope, the slightly
serrated edge of the colony is composed of long, interlacing chains of bacilli, resembling
locks of matted hair. This is referred to as medusa head or judges wig or woman’s curling
hair type of growth.
 B. licheniformis produces characteristic hair-like outgrowths from streaks of the organisms
on agar media. Colonies become brown with age. The name of this species derives from the
similarity of its colonies to lichen.
 In medium containing iron salts, virulent B.anthracis produces pink or purple colured pigment
colonies.
 Virulent capsulated strains form rough colonies, while avirulent attenuated strains form
smooth colonies.
 In gelatin stab, fine filaments of growth develop laterally along the line of inoculum. The
growth nearer to the surface of the medium is the longest and they progressively shorter.
Where there is less oxygen resembling inverted fir tree appearance.
 On haemolysis, the B.anthracis produces slight haemolysis with capsule production
compared with anthracoid organisms. B. cereus produce produce a wide zone of complete
haemolysis around the colonies.
 A selective medium (PLET) consisting of polymyxin, lysozyme, EDTA and thallous acetate
added to heart infusion agar are useful for isolate B.anthracis from mixtures containing other
spore-bearing bacilli.

BIOCHEMICAL CHARACTERS

They are Catalase +ve. It ferments Glucose, maltose, saccharose, trehalose and dextrin produce
acid but no gas. Nitrates reduced to nitrites. Indole is negative.

RESISTANCE

o
The vegetative bacilli are destroyed at 60 C in 30mts. In the carcases of animals the bacilli
remain viable in the bone marrow for a week and in the skin for two weeks. Normal heat fixation of
smears may not kill the bacilli in blood film.

The spores are highly resistant to drying, heat, cold and disinfectants. Spores remain viable
for many years in soil, water and animal hides and products. Spores have been isolated from naturally
o
infected soil as long as 60years.They resists dry heat at 140 C for 2-3hrs and boiling for 10mts. They
o
survive in 5% phenol for weeks. Spores can be killed at 120 C for 10min and 4% Kmno4 treatment for
15mts. Destruction of the spores in animal products is achieved by HCHO. Treat 2% solution of HCHO
o o
at 39-40 C for 20 mts for disinfection of wool and as 0.25% at 60 C for 6hrs for animal hair and
bristles. This process is called as duckering.

The anthrax bacillus is susceptible to sulphonamides, pencillin, erthromycin, streptomycin,

 15

tetracycline and chloramphenicol.

ANTIGENS AND TOXINS

The complex antigenic structure includes a capsular polypeptide, a somatic protein and a
somatic polysaccharide antigen. The capsule contains poly-d-glutamic acid, which is only detectable
in virulent strains, having antiphagocytic property. The somatic protein is protective antigen present in
the edema fluid.

B. anthracis produces an extracellular toxin which is composed of three components, Factor I,

II & III namely oedema factor (EF), protective antigen (PA) and lethal factor (LF) respectively. Factor I
consists of chelating compound containing phosphorus with protein and carbohydrate moieties.
Factor II & III consists of proteins. All three factors are essential to exhibit their toxic properties. They
are not toxic individually but the whole complex produces local odema and generalized shock.

OTHER TOXINS

 InH – immune inhibitor

 MprF- multiple peptide resistance factor
 Anthrolysins – similar to phospholipase C and cholestrol binding cytolysins – lysis of
phagolysosome - liberate B.anthraces- lysis of cell membrane – liberate into extra cellular
environment
 DIp- iron binding protein
 AtxA – anthrax toxin activator
 AcpA- anthrax capsule activator

PATHOGENESIS
Spores are acquired from the environment. They are phagocytosed by macrophages. The spores
germinate within the phagolysosome compartment. Vegetative bacteria produce the regulatory
proteins AtxA and AcpA, which in turn up regulate the production of LeTx, EdTx, capsule and other
toxins. InH and MprF aid in the survival of B.anthracis while it is in the phagolysosome.

Anthrolysins permit escape from first the phagolysosome and then the macrophage itself.
Production of lethal toxin triggers the hyper production of cytokines (IL-1, 6 & 8, TNF) leading to
multiple organ dysfunction, shock and depletion of clotting factors as well as apoptoptic death of
macrophages. Edema factor produces severe fluid and electrolyte imbalances, resulting in localized
odema and systemic shock.

B anthracis Bacterial Bacteremia Vascular

Contaminated forages Proliferation and multiply in &
or carcasses by capsule) (Resist phagocytosis regional LN’s toxemia
by capsule)

Permeability
is increased

Shock & asphyxia

SYMPTOMS

Horses

Acute form is very common and death may take place one day after edematous swelling of
the throat and neck region. There may be symptoms of colic. In less acute, oedmatous swelling
become generalized and death occurs after 2-3 days.

Cattle
 16

Bulls are more susceptible than cows. They have a mortality rate of 90%. There are three
clinical causes of bovine anthrax. In peracute sepeticemia death occurs within 2 hours after animal
collapsing with convulsions, sudden death in animals that appeared normal is common.

In acute septicemia death occurs within 48 to 96 hours clinical signs include fever, anorexia,
ruminal stasis, hematuria and blood tinged diarrhea. Pregnant animals may abort and milk production
often abruptly decreases. Terminal signs include severe depression, respiratory distress and
convulsions.

In chronic cases, clinical signs are manifested for more than 6 days are rare. B.licheniformis
infection is associated with the feeding of contaminated silage and is responsible for abortion in
cattle and sheep.

Sheep

Disease is acute and death occurs rapidly after convulsions.

Pigs

They have a greater natural resistance than herbivores. The usual signs are oedematous
swellings in the region of throat and neck interfering normal respiration, enteritis and rise in
termperature. The disease is not always fatal.

Dogs

The throat will be inflammed and swollen with gastroenteritis

LESIONS

The carcass of animals will putrify rapidly and develop incomplete rigor mortis. The blood is
dark, (tarry colored), clots poorly & exudes from the natural orifices. The spleen is greatly enlarged,
dark and friable. The spleen reveals black cherry jam consistency. The LN (lymph node) at the region
of initial infection site is hemorrhagic and edematous. Ecchymotic hemorrhages on the serosal
surface of the abdomen, thorax, epicardium and endocardium are common. Subcutaneous
edematous swellings are present on the ventral aspect of the neck

(Note: When suspected for anthrax care should be taken not to open the carcass. Muzzle
piece or earpiece is usually sent for examination)

Diagnosis

1. Clinical symptoms

2. Blood films from dead animals made by puncturing the superficial vein of the ear or in the region of
the foot. Care should be taken to seal the injection site by placing cotton soaked in alcohol and
ignited. The smears are heat fixed and stained by wright or giemsa’s stain to reveal B. anthracis as
large blue rods with characteristic dark pink or purple coloured capsules. In case of horse and pigs
since peripheral blood contains fewer organisms, smears should be made from the
edematous fluid or LN’s.

3. Cultural examination:

 Swabs from blood are inoculated in blood agar plates and incubated at 37°C for
24 hrs and examined for their typical growth.
 Swabs are inoculated in agar enriched with blood or serum and incubated for 6 hrs at 37°C
and examined by stained smears.

4. Bacteriological examination of hair, wool, hide, bone, bone meal & others

Sample are added to cold saline and shaken intermediately for 3 hours. The supernatant
 17

fluid is then heated to 70°C for 10 minutes. Then they are filtered through two layers of muslin cloth,
added to melted agar, and poured into petridishes, allowed to set and incubated at 37°C. After 12
hours incubation, plates examined for characteristic colony morphology.

5. Ascoli’s Precipitation test: (Ascoli 1911)

Grind up the organ or blood of suspected animal and suspend to 5-10 parts of saline and boil
for 15 minutes. Filter through filter paper and allow cooling. Place 0.5 ml of anti-anthrax serum (1:50)
in a small test tube and overlay with 0.5 ml of clear filtrate. Stand at room temperature for 15 minutes.
A white ring of precipitation indicates a positive reaction.

6. String of Pearl’s test: (Charlton, 1980)

B.anthracis produces swollen round cells in chains (string of pearls) when incubated for 3-
6hrs on tryptose agar containing 0.05 –0.5 IU of pencillin/ml

To 100ml of molten nutrient agar, add required sodium bezyl pencillin and mix carefully.
2
Pour into petridishes and allow to set. With a scalpel cut a block about 1.6 cm from the pencillin agar
plate and place it on a microscopic slide in a petridish containing a small piece of moistened
absorbent cotton wool to prevent the agar drying out. Use a young colony to streak inoculates the
0
center of the agar block. Place a clean coverslip on the agar block and incubate the petridish at 37 C.
After 2hrs, remove the slide and examine the inoculum microscopically by oil immersion for the string
of pearls growth.

7. Fluorescent antibody technique.

8. Bacteriophage identification (Differentiation from non-pathogenic Bacillus sp.)

It is considered reliable but needs experience. Cherry gamma phage is used. It can be
propagated on B. anthracis strain 14, which makes a suitable control. On one half of blood agar plate,
streak a B. anthracis strain 14 cultures as control and on the other half streak the culture to be
identified. Add drops of phage preparation (1:10 dilution) on both halves. Incubate the plates in the
upright position for 8-12 hrs. Clear zones of clearing will be seen on control culture and on the
suspected half if it is B. anthracis

9. Animal Inoculation: Guniea pigs & mice are highly susceptible. Materials are inoculated by dermal
scarification, subcutaneous or intramuscular. Death occurs in 2-3 days and the organisms will be
readily identified in the blood & tissues.

Apart from this laser pyrolysis, gas-liquid chromatography, mass spectrometry is

used for detection of anthrax toxin productions.

TREATMENT

The organism is susceptible to penicillin-G, tetracyclines, erythromycin and chloramphenicol.

IMMUNITY

Prevention of anthrax in animals is aided by active immunization. Initially oedema fluid and
tissues obtained from anthrax lesion, which had been freed from viable organisms, had protective
properties. He termed the active substances as “aggresins”. Pasteur’s vaccine was anthrax bacillus
o
attenuated by growth at 42 –43 C.

As the spore is the common infective form in nature, vaccines consisting of spores of
attenuated strains were developed. The Strene vaccine contained spores of a noncapsulated avirulent
mutant strain. It should be given 1 month before anticiopated outbreaks. The Mazucchi vaccine
contained spores of stable attenuated carbazzoo strain in 2% saponin. It gives good protection when
given subcutaneously. Protective humoral antibodies develop in 7-10 days against Factor II of the
exotoxin complex and lasts about one year.
 18

PUBLIC HEALTH ASPECTS

There is need for great care in performing necropsy on animals. Infections most often result
from spores entering through injuries to the skin causing cutaneous anthrax. Spores are present in
soil, hair, hides, wool (wool sorter’s disease-Pulmonary anthrax), faeces, milk, meat and blood
products. The skin lesion (cutaneous anthrax) is usually solitary, painless, seropurulent, necrotizing,
hemorrhagic and ulcerous. It leaves a black scar (anthrax=coal), which accounts for the name
malignant pustule.

Difference between B. anthracis & Anthracoid organisms in culture

Sl.no. B. anthracis Anthracoid organisms

1. Non motile generally motile

2. Capsulated non capsulated
3. Grows in long chains Grows in short chains
4. No turbidity in broth Turbidity in broth
5. Inverted fir tree in gelatin Atypical or absent
6. Methylene blue reduced weakly Reduced strongly
7. Haemolysis weak Strong
8. Liquefaction of gelatin is slow Rapid
9. Lecithinase reaction is weak Rapid
10. Ferments salicin slowly Rapid
11. Produces toxin neutralised by not neutralized
B. anthracis antitoxin
12. Pathogenic to g.pigs & mice non pathogenic
13. Susceptible to gamma phage not susceptible.

Difference between B. anthracis & Anthracoid organisms in blood smear stained with Polychrome
methylene blue stain

B. anthracis Anthracoid organisms

1. Dark pink or purple coloured capsules No capsule
2. Organism rod stained blue Organism rod stained blue
3. Ends truncated Ends bulged and rounded
4. Single, pair or short chain Usually long chain
5. Absence of spores Spores may be present
CLOSTRIDIUM

The genus Clostridium consists of gram positive, spore forming anaerobic bacilli. The spores
are wider than the bacillary bodies, giving the bacillus a swollen appearance, resembling a spindle.
Hence, the name clostridium (Kloster- meaning spindle) was given.

SYSTEMATICS
Domain : Bacteria
Phylum : Firmicutes
Class : Clostridia
Order : Clostridiales
Family : Clostridiaceae
Genus : Clostridium

The clostridia can be divided into four major groups according to the kind of disease they
produce. They are as follows.

1. The Histotoxic clostridium causes a variety of tissue (often muscle) infections frequently following
wounds or other trauma (eg).

Cl. chauvoei - Cattle, sheep (pigs) - Black quarter (Black leg).

 19

Cl. septicum - Cattle, - Malignant edema

- Sheep - Braxy
- Chicken - Necrotic dermatitis
Cl. novyi type A - Sheep - Big head of rams
- Cattle and Sheep - Gas gangrene
Type B - Sheep (Cattle) - Black disease (necrotic
hepatitis)
Type C - Water buffaloe - Osteomyelitis

2. Hepatotoxic clostridia produce their toxins in the liver, thus resulting in the disease Bacillary
haemoglobinuria and Black disease

Eg;
Cl. haemolyticum - Cattle, (sheep) - Bacilliary haemoglobinuria
(Cl.novyi type D)
Cl. sordellii - Cattle, Sheep, Horses - Gas gangrene.
Cl.colinum - Birds - Quail disease,
Ulcerative enteritis
Cl.piliforme - foals, laboratory animals - Tyzzer’s disease
Calves, dogs & cats (Hepatic necrosis)

3. The Enterotoxigenic clostridium produces mainly enterotoxaemia and food poisoning although they
are occasionally histotoxic (Eg).

Cl. perfringens (Cl.welchi)

Type A - Humans - Food poisoning, gas gangrene

- Lambs - Enterotoxaemic Jaundice
(Yellow lambs disease)
- Broiler chickens - Necrotic enteritis
Type B - Lambs - Lamb dysentery
(Under 3 weeks old)
Type C - Piglets, lamps, calves - Haemorrhagic enterotoxaemia
and foals. (Clostridial enteritis)
- Broiler chickens - Necrotic enteritis
- Adult sheep and goat - Struck
Type D - Sheep - Pulpy kidney disease
(except neonates)
Type E - Calves and lambs - Enterotoxaemia

4. The Neurotoxic clostridia cause the disease by the production of the potent exotoxins (Neurotoxins)
Eg.
Cl. tetani - Tetanus
Cl. botulinum - Botulism

FAMILY CHARACTERS

They are pleomorphic, rod shaped; long filaments and involution forms are common. Spore
formation occurs with varying frequency in different species. The shape and position of the spores
vary in different species. The clostridia are motile with peritrichous flagella except Cl.welchi and
Cl.tetani type VI. Cl.welchi is capsulated, while others are not.

Clostridia are anaerobic. Cl.odematiens is strict anaerobes and is dying on exposure to

oxygen. Cl.histolyticum and Cl.welchi are aerotolerent and may even grow aerobically. The clostridia
are fermentative, oxidase negative and catalase negative organisms.

A very useful media for isolation of clostridia is Robertson’s cooked meat broth. Clostridia
grow in the medium, rendering the broth turbid most species produces gas.

 Saccharolytic species turn the meat pink – Cl.odematiens, Cl.septicum,

 20

Cl.chauvoei and Cl.welchi

 Proteolytic species turn the meat black - Cl.tetani, Cl. Botulimum, and produce foul
and pervasive odour Cl.hamolyticum

 In litmus milk medium, the production of acid, clot and gas can be detected.

NEUROTOXIC CLOSTRIDIA

CLOSTRIDIUM TETANI

It is the causative organism of tetanus in Man and Animals.

HISTORY

 Tetanus has been known from very early times, having been described by Hippocrates. But
the knowledge of the disease was achieved only in 1884.
 Rosenbach –1886 - demonstrated a slender bacillus with round terminal spores in a case of
tetanus.
 Kitasato –1889 – isolated Cl.tetani in pure culture and reproduced the disease in animals by
inoculation of pure culture.
 The Greek term “tetanus” which means ‘contracture’ has been taken from the Latin medicine
“rigor”.

NATURAL HABITAT

Soil, especially that contaminated by animal faeces, is the natural habitat as Cl.tetani is often
transient in the intestines of horses and other animals. It is ubiquitous and has been recovered from a
wide variety of other sources, including street and hospital dust, cotton wool, bandages, catgut,
plaster of paris, clothing etc. It may occur as an apparently harmless contaminant in wounds.

MORPHOLOGY

Cl.tetani is a straight, slender, gram positive rod that characteristically produces terminal,
spherical endospores that bulges the cell giving the characteristic drumstick appearance. (The young
spore may be oval rather than spherical). It occurs singly and occasionally in chains. It is non-
capsulated and motile by peritrichous flagella.

CULTURAL CHARACTERS

o
Very strict anaerobe grows at an opt.temperature of 37 C and pH7.4. It grows on ordinary
media and the growth is improved by blood and serum and not by glucose.

Surface colonies are very difficult to obtain as the growth has a marked tendency to swarm
over the surface of the agar especially if the medium is moist. The swarming nature /spreading can
be inhibited by increasing the concentration of agar upto 3% (stiff agar). In this stiff agar individual
rhizoid colonies are formed.

On blood agar, it develops partially translucent, grayish colonies with filamentous edges
giving a fuzzy appearance. On horse blood agar, alpha haemolysis is produced, which later develops
into beta haemolysis due to the production of haemolysin (tetanolysin).

Cl.tetani grows well in Robertson’s cooked meat broth, with turbidity. The meat is not
digested, but is turned black on prolonged incubation. In gelatin stab cultures a fir tree type of growth
occurs, with slow liquefaction. A greenish fluorescence is produced on media containing neutral red.

BIOCHEMICAL REACTIONS
 21

Cl.tetani has feeble proteolytic, so it does not ferment any sugars. It forms indole. It is MR and
VP negative, nitrates not reduced.

RESISTANCE

The endospores are highly resistant and while boiling kills the spores of most strains in 15mts.
0 0
Autoclaving at 121 C for 15mts and dry heat temp of 150 C for more than one hour is completely
sporocidal. Spores are able to survive in soil for years and they are resistant to most antiseptics. They
are not destroyed by 5% phenol or 0.1% mercuric chloride solution in two weeks or more. Iodine (1%
aqueous solution) and H2O2 kill the spores within a few hours.

ANTIGENICITY

Ten serological types have been recognized based on the flagellar antigen (types I to X). Type
VI contains non flagellated strains. All types produce the same neurotoxin- tetanospasmin. This can
be neutralized by one common antitoxin.

They have a common heat stable somatic antigen shared by all types. A second somatic
antigen is shared by type II, IV, V and X.

TOXINS

Cl.tetani produces atleast two distinct toxins.

 Tetanospasmin
 Tetanolysin

They are antigenically and pharmacologically distinct and their production is mutually
independent.

Tetanolysin

Tetanolysin is a cholesterol binding cytolysin. It binds to cholesterol-containing rafts in the

eukaryotic cell membrane. Once bound it forms a pore resulting in the death of the cell. Tetanolysin
has no known pathogenic significance in the production of tetanus.

It causes lysis of rabbit and horse RBC’S. It is heat and oxygen labile similar to those of
streptolysin O,  toxin (Cl. Oedematiens) &  toxin (Cl. Welchii)

Tetanospamin

Tetanospasmin is a ‘di-chain’ molecule consisting of a light chain with (zinc endopeptidase

activity), a heavy chain composed of a translocation domain (responsible for forming a pore through
which the light chain passes), and a binding domain (responsible for binding to nerve cells).

Tetanospasmin is a zinc endopeptidase, which hydrolyzes the docking proteins required by

neurotransmitter-containing vesicles to fuse with the presynaptic membrane. Tetanospasmin
hydrolyzes the docking proteins VAMP- vesicle associated membrane protein, also known as
synaptobrevin.

PATHOGENESIS

Cl.tetani has little invasive power. The endospores enter traumatized tissue or surgical
wounds, especially after castration or docking, via the umbilicus or into the uterus following dystocia
in cattle and sheep. The spore implanted in a wound can germinate and multiply only if the conditions
are favourable.

Destruction and necrosis of tissue, lack of drainage in the area, presence of extraneous
matter especially of soil, all create anaerobic conditions and favour germination of Cl.tetani spores.
The resultant vegetative cells multiply at the site and produce the potent tetanospasmin. This travels
via peripheral nerves or blood stream and attaches to ganglioside receptors on the nearest
 22

cholinergic nerves and is internalized within a vesicle (by recptor mediated endocytosis). Then travels
retrograde inside the axons to the cell bodies in the ventral horns of the spinal cord thus affecting
many groups of muscles at various levels.

SYMPTOMS

It is influenced by several factors, such as the site and nature of the wound, the dose and
toxigenicity of the contaminating organism. The incubation period is variable from 2 days to several
weeks but is commonly 6-12 days.

Initial symptoms include mild stiffness and unwillingness to move. This may proceed to with
head, neck and tail becoming rigid. Mild twitching of muscles develops into obvious spasms of
muscles, which can occur in response to sudden noises, animal fall over to one side and unable to
rise.

In the terminal stages the rigidity of muscles extend from the limbs to the trunk, nostrils get
dilated, ears erect, nictitating membrane protrued and mastication becomes impossible because the
mouth cannot be opened – hence called “Lock Jaw”. Respiration becomes shallow and rapid before
final respiration failure.

LESIONS

No characteristic lesion for this disease but there may be a superficial wound which has
developed from accidental injury or from surgery.

DIAGNOSIS

In tetanus, the diagnosis is often based on the history and on the characteristic clinical signs
without reference to laboratory tests.

Direct microscopy

Demonstration of characteristic drumstick spores of Cl.tetani by gram stained smears of

material from a wound (but it is not confirmative, because Cl tetanomorphum and Cl.tetanoides also
produce drumstick spores).

Isolation

0
Necrotic tissue from a wound or wound exudates can be heated to 80 C for 20mts and used
to inoculate a blood agar plate and another blood agar plate containing stiff agar. A tube of
thioglycollate medium or cooked meat broth could also be inoculated and subcultured into blood agar.
The plates are to be incubated anaerobically for 2-3 days. Growth is noticed using a handlens as a
filamentous growth spreading through out the medium. The edges of this growth give pure culture on
subcultivation.

Confirmation is done by identification of toxin. The toxin present in animal’s serum or in

filtrate from cooked meat broth or thioglycolate medium can be inoculated in to mice S/C or I/M and
identified by neutralization or protection tests using specific antitoxin.

CONTROL AND PREVENTION

The disease is due to the action of the toxin, and hence, the obvious and most dependable
method of prevention is to build up antitoxic immunity by active immunization.

The available methods of prophylaxis are;

1. Surgical attention
2. Antibiotics
3. Immunization – passive, active or combined.

Surgical attention aims at removal of foreign bodies, necrotic tissue and blood clots, in order
 23

that an anerobic environment favourable for the tetanus bacillus is not provided. Flushing with
hydrogen peroxide in the wound area is produces aerobic conditions.

Tetanus can be prevented by antibiotics (Large doses of penicillin) when administered 4hrs
after infection but not after eight hrs. Hence, prompt administration is essential. Bacitracin or
neomycin may be applied locally. Pencillin can be given as both injections and orally till healing is
established. Antibiotics have no action on the toxin.

Antitoxin should be administered promptly, either I/V or into the subarachnoid space, on three
consecutive days to neutralize unbound toxin. Toxoid may be given subcutaneously to promote an
active immune response even on those animals, which have received antitoxin. For prevention, the
farm animals should be vaccinated routinely with tetanus toxoid.

CLOSTRIDIUM BOTULINUM

Cl. botulinum denotes a group of bacteria that produce extremely potent neurotoxins. These
toxins cause botulism, a disease characterized by flaccid paralysis in many animals and humans.
Botulism is most common in water birds, ruminants, horses, mink and poultry.

Botulism in animals has been called a variety of names;

Horses : Spinal typhus / Shaker foal syndrome
Cattle : Lamsiekte, loin disease and contagious bulbar paralysis
Water fowl : Limberneck, alkali poisoning and western duck sickness
(Note: Botulism is rare in domestic cats. Pigs and dogs are relatively resistant)

HISTORY

The name botulism is derived from sausage (botulus, latin for sausage), an article of food
that used to be associated with the type of food poisoning. Cl.botulinum was first isolated by
VanErmengam (1896) from a piece of ham that caused an outbreak of botulism.

NATURAL HABITAT

The endospores are widely distributed in soils and aquatic environment through out the world.
The disease botulism is mainly due to the ingestion of preformed toxin. Germination of the
endospores, with growth of vegetative cells and production of toxin, occurs in anaerobic situations
such as contaminated cans of meat, fish or vegetables, carcases of invertebrate and vertebrate
animals, rotting vegetation and baled silage.

MORPHOLOGY

Gram +ve, straight rods, occurring singly, pairs or occasionally in chains, Spores are oval,
wider than the bacilli, situated centrally, terminally or subterminally, Non-capsulated, motile by
peritrichous flagella.

Classification

Toxin Most susceptible Sources of toxin Disease

Type
produced animals
Humans, chickens, Vegetables, fruits, meat and Food borne botulism
A A
pigs fish
Waterfowl Invertebrate carcases, rotting Limberneck in long
Cα C1 vegetation and material on necked birds
refuse dumps
Cattle, horses, mink, Carcases, baled silage,
Cβ C2 dogs chicken manure as feed Forage poisoning
supplement
D D Eating contaminated bones
 24

and carcases of small

Cattle, sheep mammals (Phosphorus Lamsiekte
deficiency-Pica)
CULTURAL CHARACTERS

O
It is a strict anaerobe, opt.temp is 30-37 C and pH is 7-7.6. Good growth occurs on ordinary
media. Surface colonies are large, irregular, and semitransparent with fimbriate border. Spores are
O
produced consistently when grown in alkaline glucose gelatin media at 20 to 25 C.

In horse blood agar, large transparent colonies with irregular edges are developed with a
narrow zone of haemolysis. On sheep blood agar Cl.botulinum produce beta-haemolysis, the colonies
are slightly domed with a ragged edge.

In cooked meat medium, the proteolytic strains type A, B and F produces blackening of meat
and non-proteolytic strains C, D and E do not blacken meat. Gelatin liquefied rapidly by type A&B and
slowly or not at all by type C, D and E.

RESISTANCE

O O
Spores are highly resistant surviving several hours at 100 C and for upto 10mts at 120 C. But
O O
spores can be killed at 121 C for 15mts, while the toxins are destroyed at 100 C for 20mts.

ANTIGENS AND TOXINS

Cl.botulinum possesses a number of H, O and spore antigens. There are seven types of
botulinum toxin. Based on the toxin production Cl.botulinum is classified into 8 types (Type A to G).
The toxins differ from other exotoxins in that it is not released during the life of the organism. It is
appears in the medium only on the death and autolysis of the cell. It is believed to be synthesized
initially as a nontoxic protoxin or progenitor toxin. Trypsin and other enzymes activate progenitor
toxin to active toxin.

These neurotoxins are identical in pharmacological action but differ in potency, distribution
and antigenicity. They are neutralized only by the homologus antiserum. All seven types of toxins are
zinc endopeptidases. They hydrolyzes the docking proteins required by neurotransmitter-containing
vesicles to fuse with the presynaptic membrane. Though the end results is the same (blockage of the
release of neurotransmitter), the various types of toxins hydrolyze different docking proteins.

Comparison of the toxins of Cl.tetani and Cl.botulinum

Characteristics Cl.tetani Cl.botulinum

Site of toxin production Wounds Carcases, decaying vegetation and
occasionally wounds and intestine
Mode of action Centrally by blocking synaptic Peripherally by blocking neuromuscular
inhibition transmission
Type of paralysis Spastic paralysis Flaccid paralysis
Antigenic types of toxin Tetanospasmin Eight different toxins produced by types A
(one antigenic type) -G.

PATHOGENICITY

Ingested botulinum toxin absorbed from the glandular stomach and anterior small intestine
and distributed via the blood stream. It binds to receptors and enters the nerve cell after receptor
mediated endocytosis. Vesicles containing toxin remain at the myoneural junction. A light chain of
the toxin translocates across the vesicle membrane into the cytosol of the nerve cell and
subsequently hydrolyzes docking proteins. The synapse degenerates and flaccid paralysis results due
to the lack of neurotransmitter- actylcholine. When this affects muscles of respiration, death due to
respiratory failure occurs. Cl.botulinum is non invasive and virtually non infectious. Botulism is of
three types;
 25

1. Food borne botulism

It is due to the ingestion of preformed toxin in foodstuffs. The toxin is adsorbed from the
intestinal tract and is transported via the blood stream to peripheral nerve cells. Where it binds to
susceptible cells and suppresses the release of acetylcholine at the myoneural junctions. This results
in flaccid paralysis, death being caused by circulatory failure and respiratory paralysis.

2. Wound botulism

The spores are introduced into wounds where they germinate. Toxin is formed at this
localized site and spreads through the body. The shaker foal syndrome in horse is thought to be
caused in this way.

3. Infant botulism

It occurs when spores are ingested in food and get germinate in the intestines when the
normal flora has not been fully established. This form is seen in human infants (Floppy baby
syndrome) and as rare epidemics of type C in broiler chickens and turkey poults.

SYMPTOMS

In cattle, the incubation period varies between 2-10 days depending upon the dose of toxin
ingested. Initially there will be excitation, followed by incoordination and paralysis of the hind limbs.
There will be paralysis of muscles in the mouth, pharynx and neck resulting in the animal being unable
to swallow and the tongue protruding from the mouth. This is followed by death. In South Africa this
condition is termed as lamsiekte in cattle caused by type D especially in the phosphours deficient
animals.

In poultry it results with the ingestion of type C toxin and the disease is known as duck
sickness or western duck disease and Limberneck (drooping head posture) in chicken. The
symptoms include paralysis of the wings, legs and neck, protrusion of nictitating membrane, diarrhea
and comatase before recovering in 5-6 days time.

In horses the incuation period varies between 12 hours to ten days. The symptoms include
generalized muscle weakness, slow eating and a shuffling gait with toe dragging. Sluggish pupillary
light response may be detected within 6 to 18 hours after toxin ingestions. The tongue tone, eyelid
and tail tone are weak.Prehension of food and ability to swallow are usually affected in horses with
botulism. Affected one walk with a stiff, stilted gait and short “choppy” strides and oftentimes have
muscle fasciculations progress to recumbency within 12-24 hours

LESIONS

rd
Pathological changes in the CNS especially the brain stem and 3 ventricle, catarrhal
gastroenteritis, hepatitis and nephrosis

DIAGNOSIS

The diagnosis of botulism is based on history, clinical signs and demonstration and
identification of toxin in serum of moribund or recently dead animals as well as the detection of toxin
and /or Cl.botulinum in the suspected foodstuff.

Toxin demonstration

Serum or centrifiuged serum exudates from animals can be directly inoculated I/V (0.3 ml) or
I/P (0.5 ml) into mice. If toxin is present the characteristic wasp waist appearance in the mice will be
seen in a few hrs or upto 5 days. The appearance is due to abdominal breathing because of paralysis
of respiratory muscles.

Extraction of toxin in foodstuffs is accomplished by grinding the material in saline. The

 26

suspension is centrifuged and the supernatant is filtered through a 0.45μm filter. As the toxin can be
in a protoxin form 9 parts of filtrate are treated with one part of 1%trypsin solution and incubated at
0
37 C for 45mts. Mice or guinea pigs are inoculated intra-peritoneally.

Toxin identification

Mouse (or guinea pig) neutralization test using a polyvalent antitoxin initially followed by
monovalent antitoxin is used to identify the toxin.

Isolation of Cl.botulinum from foodstuffs

Several samples of the foodstuffs are macerated in a small amount of physiological saline.
0
The suspension is heated at 65-80 C for 30mts to kill most of the contaminating organism and to
induce the Cl.botulinum spores to germinate. Blood agar plates are inoculated with the suspension
0
and incubated under CO2 at 35 C for upto 5 days. To determine whether the isolate is a toxin
0
producing strain, a cooked meat broth is inoculated and incubated at 30 C for 5-10 days. Filtrates are
prepared and lab. Animals can be used for demonstrate and identification of the toxin.

TREATMENT

Neutralization by polyvalent antiserum is effective. Therapeutic agents such as

tetraethylamide and guanidine hydrochloride, which enhance transmitter release at neuromuscular
junctions, may be of value when given intravenously.

Control and Prevention

Immunization is not followed. In South Africa attempts were made by giving two injections of
types C&D toxoid at an interval of several weeks. Bivalent & Trivalent antitoxins are available
commercially.

CLOSTRIDIUM PERFRINGENS (CLOTRIDIUM WELCHI)

HISTORY

The bacillus was originally cultivated by Achalme (1891), but it was first described in detail by
Welch and Nuttal (1892)-who isolated it from the blood and organs of cadaver.

NATURAL HABITAT

Type A occurs in the intestinal tract of human and animals and in most soils. Type B to E is
more adapted to survival in the intestines but in outbreaks of disease they survive long enough in soil
to infect other animals.

MORPHOLOGY

Gram +ve, bacillus, straight, parallel sides, rounded or truncated ends, occur either in singly or
in chains or small bundles. It is highly pleomorphic, filamentous and involusion forms are common. It
is capsulated and non-motile. The spores are oval, subterminal and bulge the mother cell. They are
rarely produced and their absence is one of the characteristic morphological features of Cl.welchi.

CLASSIFICATION

Based on the type of toxins, they are classified into 5 types

Cl.perfringens Major toxins Host Disease

types
 27

Enterotoxins Human Food poisoning

A Alpha Lambs Enterotoxaemic jaundice
Broiler chickens Necrotic enteritis
Beta and alpha Lambs under 3 Lamb dysentery
B
weeks old
Piglets 1-3days old Haemorrhagic enteritis
(Clostridial enteritis)
Beta and alpha Broiler chickens Necrotic enteritis
C
(2weeks old)
Adult sheep and Struck
goats
Epsilon Sheep all ages Pulpy kidney disease
D
(except neonates) (over eating disease)
Iota and alpha Calves and lambs Enterotoxaemia (Haemorrhagic
E
enteritis)

CULTURAL CHARACTERS

It is an anaerobe, but can also grow under micro aerophilic conditions. Oxygen is not actively
0
toxic to the bacillus and cultures do not die on exposure to air. Though, this bacillus is grown at 37 C,
0
pH 5.5-8.0, the temp of 45 C is optimal for many strains. The generation time at this temperature is 10
minutes only. This property can be utilized for obtaining pure cultures of Cl.welchi. Robertsons
0
cooked meat broth inoculated with mixtures of Cl.welchi and other bacteria and incubated at 45 C for
4-6hrs serves as enrichment. Subcultures from this onto blood agar plates yield pure or predominant
growth of Cl.welchi

Good growth occurs in Robertson’s cooked meat medium. The meat is turned pink but it is
not digested. In litmus milk, fermentation of lactose leads to formation of acid, which is indicated by
the change in the color of litmus from blue to red. The acid coagulates the casein (acid clot) and the
clotted milk is disturbed due to the vigorous gas production. The paraffin plug is pushed up and
shreds of clot are seen sticking to the sides of the tube. This is known as stormy fermentation.

After overnight incubation on rabbit or sheep blood agar, colonies of most strains show a
target haemolysis resulting from a narrow zone of complete haemolysis due to theta toxin and a
much wider zone of incomplete haemolysis due to alpha toxin. This double zone haemolysis pattern
is characteristic for Clostridium welchi. All the five types of Cl.welchi (A –E) produce alpha toxin. This
is a lecithinase C, which, in the presence of calcium and magnesium ions splits lecithin into
phosphotidyl choline and diglyceride. This specific lecithinase effect can be demonstrated by Nagler’s
reaction.

Type A antitoxin is spread over half of an egg yolk agar plate and allowed to dry. The suspect
Cl.perfringens is streaked across both sides of the plate. All the types of Cl.perfringens produce the
alpha toxin that is a lecithinase. On the half of the plate without the antitoxin, the lecithin in the
medium is attacked causing opalescence around the streak. The lecithinase reaction is neutralized on
the other half of the plate with the antitoxin but the growth of Cl.perfringens is unaffected.

BIOCHEMICAL REACTIONS

Cl.welchi ferments several sugars (glucose, maltose, lactose and sucrose) and produced acid
and gas. Indole –ve, MR +ve, VP-ve, H2S +ve.

RESISTANCE

Spores are usually destroyed within 5minutes by boiling, but those of the food poisoning
strains of type A and type C strains resist boiling for 1-3 hrs. Autoclaving temp is lethal.

TOXINS
 28

Cl.welchi is one of the most prolific of toxin producing bacteria forming at least 12 different
toxins, besides many other enzymes and biological active substances.

Cl. perfringens Major toxins

Type Alpha Beta Epsilon Iota
A + - - -
B + + + -
C + + - -
D + - + -
E + - - +

Alpha toxin:

Alpha toxin is produced by all types. Mostly by type A strains. It is lethal, dermonecrotic and
haemolytic. This is a lecithinase C (phospholipase) that attacks cell membranes causing cell death
and destruction and also responsible for Nagler’s reaction. It hydrolyzes phosphotidylcholine and
sphingomyelin, both of which are constituents of host cell membrane.

It is haemolytic for the red cells of most species except horse and goat. This toxin gives a
zone of partial haemolysis on blood agar. The haemolysis is of hot-cold variety being best seen after
0 0
incubation at 37 C followed by chilling at 4 C.

Beta toxin:

Beta toxin is lethal and necrotising. It is a pore forming toxin, which damages host target cells
such as intestinal epithelial cells and endothelial cells. It is sensitive to trypsin and this explains the
predlection of types B and C for neonates as colostrum has anti trypsin activity. It is a labile toxin and
may be destroyed if there is a delay in small intestinal contents, containing the toxin, reaching the
laboratory.

Epsilon toxin:

Epsilon toxin is secreted as a protoxin (proto toxin) and is activated in the intestines by
proteases such as trypsin. Pulpy kidney disease is not usually seen in neonatal lambs as colostrum
contains an antitrypsin factor that can prevent the epsilon toxin being activated. The toxin itself
increases gut permeability, assuring absorption of the toxin into the blood stream. It damages
vascular endothelium (including blood vessels in the brain) leading to fluid loss and odema. This
epsilon toxin can be regarded as an enterotoxin and neurotoxin.

Iota toxin:

Iota toxin is also produced as a protoxin and is not unique to Cl.perfringens type E as it is also
formed by Cl.spiroforme and Cl.difficile.It is a binary toxin composed of a binding portion that binds
the toxin to target epithelial cells, and an enzymatically active portion. It is also a pore forming toxin.

Perfringolysin O (also known as theta toxin):

It binds to cholesterol containing rafts in the eukaryotic cell membrane, and forms a pore,
which results in the death of the cell. It is also responsible for lysis of phagolysosomal membrane.

Enterotoxin:

It is produced during sporulation of Cl.perfringens. When the endospore is released,

enterotoxin is also released in the surrounding media. It is bifunctional toxin, first forming a pore in
the apical portion of small intestinal epithelial cells resulting in fluid and electrolyte abnormalities, as
well as providing access to tight junction proteins results in further losses in control of fluid and
electrolytes.

Besides several minor toxins are produced such as the haemolysin, Kappa (collagenase),
lambda (Proteinase), Mu(hyaluronidase) and Nu(DNase) – all these may contribute to tissue damage.
 29

PATHOGENICITY

The enterotoxemias are often precipitated by certain husbandry and environmental factors
such as abrupt changes in feeding usually to a richer diet and overeating and voracity on high protein
and energy rich feeds. This leads to slowing of peristalsis with retention of bacteria in the intestines,
absorption of toxins, inadequately digested carbohydrate and the provision of a rich medium for the
proliferation of Cl.perfringens

The bacterium inhabits the large intestine in normal animals, but if overgrowth occurs
Cl.perfringens can spill over into the small intestine with the production of a large amount of toxin and
enterotoxaemia

Cl.perfringens Type A:

Tissue destruction is probably due to the membrane active toxins (alpha and perfringolysin O),
and the toxins that affect connective tissue (collagenase, hyaluronidase, and sialidase)

Cl.perfringens Type B:

Beta toxin is considered the principal factor producing hemorrhagic enteritis affecting the
small intestine. In lambs the signs are depression, anorexia, abdominal pain and diarrhea. The course
is rapid with mortality rate approaching 100%. A chronic form occurs in older animals. The
characteristic intestinal lesion is haemorrhagic enteritis.

Epsilon toxin being a permease increases intestinal permeability, ensuring its absorption into
the circulation where it affects vascular endothelium, leading to fluid loss and odema, as well as
damage to kidney function. Congestion, odema, serosal effusions and haemorrhages in various
organs are seen.

Beta and epsilon also affect the nervous system which leads to severe depression and high
mortality.

Cl.perfringens Type C:

Beta toxin is considered the principal factor producing hemorrhagic enteritis affecting the
small intestine. In neonates the signs are depression, anorexia, abdominal pain and diarrhea. The
course is rapid with mortality rate approaching 100%. The characteristic intestinal lesion is
haemorrhagic enteritis. The signs and lesions are mainly due to alpha, beta and perfringolysin O.

Cl.perfringens Type D:

The key epsilon toxin is secreted as protoxin activated by intestinal proteases. Epsilon toxin
increases intestinal permeability, ensuring its absorption into the circulation where it damages
vascular endothelium, leading to fluid loss and odema. When toxin levels are high, affected capillary
endothelial cells in the brain are damaged, and the resultant odema greately increases the intracranial
pressure. When the amount of toxin is lower, it damages the capillary endothelial cells in the brain
leads to focal symmetrical encephalomalacia. Epsilon toxin also causes cAMP related hyperglycemia
and glycosuria.

It is common in suckling lambs 3 to 10 weeks old and in feedlot animals. Lambs may die
without premonitory signs. Gross lesions may be absent. Convulsions and diarrhea may present.
Cattle and older sheep show neural manifestations. Affected sheep display blindness, head pressing
and anorexia. In goats, diarrhea is common. Catarrhal, fibrinous or haemorrhagic enterocolitis is a
consistent lesion in goats. Death rates are high in lambs.

Disease Clinical and postmortem signs

Food poisoning Sudden onset, diarrhoea, abdominal pain and nausea. But
vomiting is uncommon. Short course and rarely fatal
Enterotoxaemic jaundice Depression, anaemia, icterus, haemoglobinuria and lambs die
 30

within 6-12hrs of first signs known as the yellows or yellow

lamb disease
Lamb dysentery A haemorrhagic and rapidly fatal enterotoxaemia. Lambs are
often found dead
Haemorrhagic enterotoxaemia Dysentry, collapse and death. Small intestine is dark red and
(Clostridial enteritis) has gas bubbles in mucousa
Necrotic enteritis in broilers Depression, diarrhoea, death in a few hrs. Mortality 2-50%.
Mucousa of small intestine has a brown psudomembrane.
Most common in deep litter units.
Struck Sudden death due to an enteotoxaemia
Pulpy kidney disease Odema of brain, glycosuria, sudden death. Excess fluid in body
(Over eating disease) cavity, focal symmetrical encephalomalacia occurs in well-
grown lambs.

DIAGNOSIS

Gram stained smears can be made from the mucousa of the small intestine of a recently
dead animal. Large numbers of gram-positive rods are suggestive of Cl.welchi.

Histopathology on brain sections helps to demonstrate focal symmetrical enceohalomalacia

in pulpy kidney disease. Rapid kidney autolysis, pulpy cortical softening and Glucosuria are suggestive
of pulpy kidney disease.

Demonstration of toxin in the small intestine

Collect 20-30ml of ileal contents from a recently dead animal and send it to the laboratory as
soon as possible. The ileal contents are centrifuged and the clear supernatant is tested for toxin. In
ileal contents, the epsilon and iota toxins are usually in the active form.
To demonstrate the toxin 0.4ml of the clarified ileal contents can be inoculated intravenously
into mice. If a mouse die within 5mts this is probably due to shock, death from toxin usually occurs
within 10hrs. Identification of toxin in the clarified ileal content is carried out by a neutralization test
by using suitable antitoxin.

CONTROL AND PREVENTION

Before the lambing season the ewes are vaccinated with formalised whole culture or alum
precipitated vaccine. The resulting passive immunity, which unweaned lambs, derived from
colostrum protect, lambs for first 3 weeks of life.

Similarly alum precipitated trypsin –treated toxoid is also satisfactory. Lambs can also be
vaccinated by giving the first dose within 72 hrs of birth and repeated at 4 weeks of age. Immunity
may not last more than 6-12 months unless booster dose is given.

HISTOTOXIC CLOSTRIDIA

Histotoxic clostridial organisms typically are involved in infections of muscle, liver or heart.

Cl. chauvoei - Cattle, sheep (pigs) - Black quarter (Black leg)

Cl. septicum - Cattle, - Malignant edema

- Sheep - Braxy
- Chicken - Necrotic dermatitis
Cl. novyi type A - Sheep - Big head of rams
- Cattle and Sheep - Gas gangrene
Type B - Sheep (Cattle) - Black disease
(Infectious necrotic hepatitis)
Type C - Water buffaloe - Osteomyelitis
Type D - Cattle, (sheep) - Bacilliary haemoglobinuria
 31

(Cl. haemolyticum)
Cl. sordellii - Cattle, Sheep, Horses - Gas gangrene
Cl.colinum - Birds - Quail disease
Ulcerative enteritis
Cl. piliforme - foals, laboratory animals - Tyzzer’s disease
Calves, dogs& cats (Hepatic necrosis)

CLOSTRIDUM NOVYI (CLOSTRIDUM ODEMATIENS)

NATURAL HABITAT

Clostridum novyi is world wide in distribution. The principal habitat of Cl.novyi is soil and the
intestine of animals. Type A commonly found in soil. Type B is rarely found in soil, and it is common in
the normal intestinal tract of herbivores. Strains of type A and B are recovered from the livers of
normal animal. Type D (Cl. haemolyticum) - is found in the ruminant digestive tract, liver and in the soil.
Based on toxin production Clostridum novyi classified into four types (A-D).

MORPHOLOGY

Large, pleomorphic, gram+ve rods with oval to cylindrical subterminal spores. There is little or
no swelling of the mother cell, non-capsulated, motile by peritrichous flagella.

CULTURAL CHARACTERS

Clostridium novyi type B and Cl.haemolyticum are very demanding in both their anaerobic and
nutritional requirements. Very strict anaerobic procedures are necessary and media containing
cysteine should be used. These clostridia can die within 15mts of being exposed to atmospheric O2

These organisms difficult to grow on primary culture and the growth are enhanced by agar
enriched with glucose or freshly prepared blood or fresh brain infusions. On blood agar Cl.novyi
produces characteristic large, irregular colonies with a rhizoid edge and a large zone of clear
haemolysis.

On moist surface of solid media after 3-4 days incubation colonial motility develops which is
characterized by the movement of daughter colonies moving away from parent colonies in spirals or
arc and few are return and fuse to the parent colony.

In horse blood agar, the colonies are haemolytic, small and usually rhizoidal in nature. Areas
of hemolytic develop beneath the colonies and develop into wider zone after 48-72hrs incubation.

In Sheep blood agar very slight haemolysis develops. In Robertson’s cooked meat medium
Cl.novyi type D is very strong proteolytic. Type A, B and C are saccharolytic. The lecithinase activity of
beta toxin of type B and D, and Gamma toxin of type A are producing quite distinct opacity changes
on egg-yolk agar.

Cl.novyi type A exhibits lipase activity on egg-yolk agar. It will produce characteristic
iridescent pearly layer on the surface of the colonies extending on to the surface of the medium
immediately surrounding them. Cl.novyi type A is the only species among clostridia that produces
both a lecithinase and a lipase.

BIOCHEMICAL CHARACTERS

Saccharolytic type ferment glucose and maltose but not lactose.

RESISTANCE

0
Spores of most strains survive heating to 95 C for 15minutes, and are killed at the autoclaving
temperature. Spores are resistant to 5% phenol, 10% formalin or 0.1% merthiolate. They are killed
 32

rapidly by exposure to hypochlorite. Spores remain viable for years in soil.

ANTIGENS AND TOXINS

They possess several somatic and flagellar antigens, which are not of much importance.
Based on toxin production Cl.novyi classified into 4 types. Cl.novyi synthesizes five major toxins, α, β,
γ, δ and ε.

Type of organisms Alpha Beta

Cl. novyi type A + -
Type B + +
Type C - -
Type D
- +++
Cl. haemolyticum

Type A produces all toxins except beta. Type C isolates are non toxigenic. In addition to this type
B also produce zeta, eta and theta toxin.
 Alpha toxin produced by type A and B is lethal, necrotizing, causes increased capillary
permeability, and is toxic to several tissues including muscle, heart and liver.
 Beta toxin is a lecithinase that hydrolyzes phosphotidyl choline and sphingomyelin and it is
produced by type B and by Cl.haemolyticum (type D) in greater amounts. This may account
for the haemolytic crisis and death in bacillary haemoglobinuria.
 Gamma toxin is a necrotising phospholipase D.
 Delta toxin is an oxygen labile haemolysin - Novyilysin
 Epsilon toxin is a lipolytic enzyme.
 Zeta toxin is partly haemolytic.
 Theta toxin is a lipase.
 Eta toxin is a tropomyosinase, which degrades tropomyosin and myosin and may play a role
in destruction of infected muscles.

PATHOGENESIS

In black disease (infectious necrotic hepatitis) and bacillary haemoglobinuria, the spores,
normally present in the intestine, may reach the liver and remain dormant in the kupffer cells. Any
destruction of liver tissue could be the inciting factor. The tissue damage is usually due to migration
of immature liver fluke (Fasciola hepatica), and anerobic conditions permits germination of spores,
growth of vegetative cells and subsequent production of toxins (alpha and beta).

Alpha toxin produced, in the local area of necrosis, in the liver is adsorbed into the circulation
and results in systemic effects. Death may be sudden or within 2 days. Signs include depression,
anorexia and hypothermia. Necropsy reveals odema, serosal effusion and liver necrosis.
Subcutaneous venous congestion secondary to pericardial odema darkens the underside of the skin,
suggesting the name black disease In case of bacillary haemoglobinuria the dominant toxin is beta
toxin (phospholipase C). It causes haemolytic crisis and death within hours. Big head in rams develop
when s/c tissues traumatized during fights and are subsequently invaded by Cl.novyi type A. Toxic
endothelial damage (by alpha toxin and novyilysin) produces odema involving head, neck and cranial
thorax. Death occurs in 2 days. The yellow tinge of the odema fluid which is clear and gelatinous with
little hemorrhage is a postmortem change.

SYMPTOMS

Big head:

Odematous swelling occurs in head, face and neck. It will be followed by collapse and death
of animals. The mortality rate may be more than 90%.

Black disease:

Acute toxaemia is leads to sudden death. The signs include rapidly /decreasing ability to
 33

move, unsteady gait and collapse.

Bacillary haemoglobinuria:

Common in summer months, affected animals suffers from fever, pale, icteric mucous
membranes, anorexia, agalactia, abdominal pain, port-wine coloured urine, diarrhea, haemoglobinuira
and hyperpnea. The mortality rate is 90%.

LESIONS

Black disease:

Number of clearly defined gray-yellow foci (necrotic areas) in the liver. The lesion consists of
a central core of necrosis surrounded by a zone of leucocytes in which there will be masses of
Cl.novyi. Excess fluid in body cavities. Straw-coloured exudates will be present in pericardial and
peritoneal cavities.

Extensive subcutaneous and bloodstained odema can be noticed in the carcass. Venous
congestion occurs that darkens the skin. (Black disease)

Bacillary haemoglobinuria:

There are number of typical anaemic infarcts in the liver. Pale and raised surrounded by a blue
-red zone. There will be blood stained intestinal contents, dark colored urine in the bladder, marked
icterus of the carcase, widespread odema and haemorrhages in the myocardium.

DIAGNOSIS

1. Based on history

2. Direct gram stained smears

Presence of characteristic liver lesions together with large number of gram +ve rods in liver
impression smears from a recently dead animal is suggestive of the disease.

3. FAT is useful for the identification of Cl.novyi type B and Cl.haemolyticum in acetone fixed liver
impression smears.

4. Isolation of organism from affected tissue (as like other clostridial infections) and by characteristic
cultural characters.

5. Animal inoculation

Toxin in the liver can be demonstrated by i/m injection of homogenates into guinea pigs. The
pathogenicity is enhanced if the homogenate is added to an equal amount of 5% Cacl2 solution before
inoculation. The guinea pigs die in 1-2 days with very extensive subcutaneous edema. Specific
antitoxin is not readily available for neutralization tests.

CONTROL AND PREVENTION

1. Elimination of liver flukes through destruction of snail

2. Aluminum hydroxide adsorbed formalized whole culture vaccines are available.

3. Outbreaks of the disease may be controlled by the prompt injection of hyper immune sera.

CLOSTRIDUM SEPTICUM
 34

Clostridum septicum is very closely related with Clostridum chauvoei, hence it is called as
Clostridum chauvoei type A.

Clostridum septicum causes

- Malignant odema in Cattle, sheep and pigs
- Braxy (Bradsot) in Sheep
- Necrotic dermatitis in Chickens
HISTORY

The bacillus was first described by Pasteur and Jourbert (1887) and named it as Vibrion
septique.

NORMAL HABITAT

It is found in soil and the intestine of animals

MORPHOLOGY

Gram +ve, highly pleomorphic, chracteristic long filamentous forms are seen in stained
smears of affected muscle. Cigar shaped rods and citron forms are more common. Spores are oval,
central or subterminal. Non-capsulated, motile by peritrichous flagella.

CULTURAL CHARACTERS

0
Strict anaerobe, growth at an opt.temp of 37 C, growth is promoted by glucose. On ordinary
media, the colonies are irregular and transparent initially, turning opaque (large, grayish white on
continued incubation). The colonies are swarming and spreading over the entire surface.

On stiff agar, the colonies are irregular with a rhizoid edge. Some strains produce smooth,
round colonies.

In cooked meat medium meat turns pink with rancid odour, and produces abundant gas
(because, it is saccharolytic). Like, Cl.perfrigens, the Cl.septicum inoculated into litmus milk produces
the classical stormy clot or stormy fermentation reaction.

BIOCHEMICAL REACTIONS

Ferment glucose, lactose, maltose and salicin but not sucrose. Acid and gas are not produced.

RESISTANCE

Spores are killed by steam in 40-50 mts and by autoclaving. Spores are also susceptible to 3%
formalin for 15mts.

ANTIGENS AND TOXINS

 Six groups have been recognized, based on somatic and flagellar antigens. Clostridum
septicum produces at least four distinct toxins and fibrinolysin.
 The α toxin is oxygen stable haemolysin, dermonecrotic and lethal. It is a pore forming toxin.
The cell bound proteolytic enzyme, furin, cleaves it, resulting in fragments that insert into the
membrane forming pores leading to death of the cell.
 The β toxin is leucotoxic and DNAse
 The γ toxin is a hyaluronidase
 The δ toxin is an oxygen labile haemolysin- speticolysin O
 α toxin has direct effect on cardiac muscle and is capable of causing capillary damage. Iron is
required both for growth of bacteria and for production of α toxin

PATHOGENESIS
 35

Braxy or Bradsot in sheep

The disease is more common in winter months. When ingestion of large volume of frozen
grass, the spores that are present in the soil are ingested with feed. The mucousa of the abomasum
is damaged due to cold conditions from an adjacent rumen. Any Cl. septicum spores present can
germinate and replication of the bacterium leads to toxin production, toxaemia and rapid death.

Malignant odema (Anaerobic cellulites)

In this exogenous gas gangrene infection, spores are introduced into wounds where they may
germinate in the anaerobic necrotic material and toxin is produced by the vegetative cells.

SYMPTOMS

Braxy usually occurs in well-nourished one-year-old sheep, ailing animals shows signs of
abdominal pain and diarrhoea. Death occurs within few hours.

In malignat odema, infected wound, which become gas gangrenous. Fever, soft swelling
around wound and spreading to muscles. Swelling odematous and wet with much exudates and gas.
Muscles appear dark red to black color.

LESIONS

In braxy, the lesion may be confined to the abomasums; there will be the characteristic area
of haemorrhagic inflammation in the wall of the abomasum. There will be an extensive quantity of
blood stained fluid in the peritoneal cavity.

DIAGNOSIS

Based on history, symptoms and characteristic lesions are strongly suggestive of this disease.

FAT is to be employed for differentiate it from Cl.chauvoei

Identification of specific Clostridial toxin by mixing 1.2ml of culture fluid with 0.3ml of
0
specific antitoxin, allowing the mixture to stand for 30minutes at 37 C and inoculate two guinea pigs
i/peritoneally. If specific toxin is present, it will be neutralized.

CONTROL AND PREVENTION

Penicillin alone or with hyper immune serum can be used to treat infections. Sheep can be
effectively immunized against the disease and multi component vaccine is used.

CLOSTRIDIUM CHAUVOEI

SYNONYMS:

 Cl. chouvoei type B, Cl. feseri

 It causes black quarter or black leg in Cattle & Sheep

NATURAL HABITAT:

Worldwide distribution in soil and pastures

MORPHOLOGY

Gram positive, rod shaped with rounded ends 3-8m in length & 5m in width. Sometimes
pleomorphic, large cigar shaped rods or citron forms occur. Non-capsulated and motile by
 36

peritrichous flagella. Non-motile variants do occur. Spores are oval and located centrally or
subterminally. Old cultures stain gram negative.

CULTURAL CHARACTERS

0
It is a strict anaerobe. Growth occurs at an optimum temperature of 37 C. Growth enhanced
by the addition of liver extract or glucose. In blood agar whitish grey colonies with irregular edges
develop surrounded by a zone of haemolysis. In cooked meat medium growth is slow and meat is
turned pink with sour odour

BIOCHEMICAL CHARACTERS

Cl. chauvoei ferments glucose, lactose, sucrose, maltose with acid & gas, but not salicin.

Resistance:

Similar to Clostridium septicum

ANTIGENS AND TOXINS

Cl. chauvoei has somatic and flagellar antigens and produces 4 toxins

Alpha toxin - Oxygen stable haemolysin, pore forming necrotoxin that causes dermonecrosis and
fibrinolysis

Beta toxin – DNA ase

Gamma toxin – hyaluronidase

Delta toxin – Oxygen – labile haemolysin. This toxin is lethal for mice & Guinea pigs when given I/v.

Chauveolysin- it binds to cholesterol containing rafts in th cell membrane. Once bound, it forms a
pore resulting in the death of the cell.

Neuraminidase – Sialidase- removes sialic acid residues from glycoconjugates on cell walls resulting
in disruptions of the intercellular matrix.

PATHOGENESIS

Ingested Cl. chauvoei spores

Remain dormant in tissues

Any trauma & damage to tissue

Latent spores in muscle become activated

Org. multiplies & produces toxins

 toxins
Dermonecrosis, myositis and fibrinolysis

Black Quarter

SYMPTOMS

The disease usually occurs in young cattle of 6 months to about 2-3 years of age. High fever,
anorexia and lameness are common. The most obvious sign is crepitate swelling particularly in the
 37

hind or fore quarter which rackels when rubbed with the fingers as a result of gas production.

The affected animal will become lame and the affected muscles shows trembling with violent
twitching. Death usually occurs within 24 hours. In sheep an acute febrile condition develops within 1-
2 days following an injury and a typical black quarter lesion can be observed at the site. Death
occurs suddenly

LESIONS

In the central part of the lesion there is usually a well-defined area of muscle, which is dark
red in colour, dry, necrotic and filled with small gas bubbles (emphysematous), which give a swollen
appearance to the muscles. The lesion has a characteristic rancid butter odour. Surrounded this area
of muscle there will be yellowish or blood stained oedematous fluid. In ewes there will be necrosis of
the vaginal mucosa and skin with extensive oedema involving the hind limbs and thigh muscles.

DIAGNOSIS

1) History
2) Symptoms
3) Smears prepared from the lesions and oedmatous fluid reveal gram positve rod.
4) Isolation can be done from the center of the lesion, oedematous fluid and from heart blood &
spleen.
5) FAT used to differentiate from Cl. septicum
6) Broth cultures or oedematous fluid from the lesions can be tested for toxicity and specific
neutralization by antitoxin in mice or Guinea pigs.

CONTROL AND PREVENTION

The most reliable results are obtained from using a formalized alum precipitated whole
culture that confers immunity against the bacteria as well as the toxin. For economic reasons a multi
component vaccine containing Cl. chauvoei, Cl. septicum, Cl.welchii type D and Cl. tetani is used.
A stronger immunity is stimulated by two doses of vaccine at a time interval of at least 2-3
weeks. Hyper immune serum (HIS) is used to control explosive outbreaks. Penicillin along with HIS is
used to treat the disease. Oxytetracycline & Chlortetracycline can also be employed effectively in early
stages.
LISTERIA

Listeria has been divided into seven species with two distinct groups. Among which the
Listeria monocyotgenes and Listeria ivanovii are haemolytic and pathogenic for animals. The Listeria
murrayi and Listeria grayi are nonhaemolytic, rarely isolated and considered to be non pathogenic.
Among which the genus L. monocytogens, being the cause of septicaemia, abortion and CNS
infections in a wide range of animal species including humans.

Host and disease syndrome of pathogenic listerias

Species Host(s) Disease

Young animals of many species, including Visceral listeriosis (septicaemic)
lambs and calves. Birds can be affected Myocardial degeneration in birds
Sheep, goats, cattle Neural listeriosis- circling disease
L.monocyotgens Sheep, goats, cattle Abortion
Cattle Iritis (Endopthalmitis)
L.ivanovii Sheep and cattle Abortion

HISTORY

L.monocytogenes first described by Murray (1926) who named it as bacterium

monocytogenes because of characteristic monocytosis infection in laboratory animals. It was
renamed Listerella hepatolytica by Pirie (1927) and the present name given by him in 1940. The
 38

Listeria momnocytogenes was first isolated by Gill (1929) from sheep.

NATURAL HABITAT

Listeria species are widely distributed in the environment and can be isolated from soil,
faeces, plants, decaying vegetation and silage (pH 5.5) in which the bacteria can multiply. Silage is
commonly implicated in outbreaks of listeriosis in cattle and sheep. In poor quality silage the listerial
7
numbers may reach 10 cfu/kg of silage. Asymptomatic faecal carriers occur in man and many animal
species. L.monocytogens can be excreted in bovine milk. Human foods associated with listeriosis in
man include soft cheeses, milk and poultry meat.

MORPHOLOGY

L.monocytogenes are medium sized, G+ve rods, non-spore forming and non-acid fast. Old
cultures stain gram –ve. From rapidly growing cultures or animal tissues the cells can appear coccal.
They are multiple by few (1-5) peritrichous flagella. They are motile at room temperature, but not at
0
37 C.

CULTURAL CHARACTERS

0
L.monocytogenes is able to grow at temperature ranges from 4 to 45 C and grow at pH range
of 5.5 to 9.6. It is relatively resistant to high salt (10%) concentrations. They are facultative anaerobes.
The growth is enhaced by agar enriched with glucose, blood, liver extract and by 10% CO2. They grow
on nutrient agar, blood agar but not on MacConkey agar. Small transparent colonies with smooth
borders appear on blood agar in 24hrs, becoming grayish white in 48hrs.

L.monocytogenes and other non-pathogenic listeria produce narrow zones of beta

haemolysis, often only under the colony itself. L.ivanovii produces a comparatively wide zone of
haemolysis and is very similar in appearance to beta haemolytic streptococci. L.monocytogenes
produces a CAMP reaction with the haemolysis of Staphy.aureus. In contrast; Listeria ivanovii is
negative in the CAMP reaction with Staphy.aureus.

Oxford medium or PALCAM medium can be used as selective media. Oxford medium contains
lithium chloride, acriflavin, colistin sulphate, cefotetan, cycloheximide, phosphomycin, ferrous ions
and aesculin. Listeria hydrolyses aesculin to aesculetin and dextrose. Aesculetin reacts with ferric
ions and produces black zones under and around the colonies. Small black, grey or brown colonies
that are surrounded by a black zone is a typical colony morphology of Listeria in this agar plate.

PALCAM medium contains Polymyxin-Acriflavin-Lithium chloride-Ceftazimide- Aesculin –

Mannitol. Listeria hydrolyses aesculin to aesculetin and dextrose. Aesculetin reacts with ammonium
ferric citrate and forms a brown-black complex seen as a black halo around grey-green colonies. On
TSA or BHI agar, these colonies have a characteristic blue-green sheen when light is reflected
0
obliquely at a 45 angle off their surface. In fluid medium, slimy tenacious precipitate forms after
incubation for several days.

L.monocytogenes, particularly shows the characteristic tumbling motility when a 2-4hr broth
0
culture, incubated at 25 C, is examined by the hanging drop method. This motility is an end-over-end
tumbling of individual cells with periods of quiescence. When grown in semisolid motility media the
Listeria spp. give an unusual umbrella shaped growth in the subsurface.

BIOCHEMICAL CHARACTERS

All the listeria spp hydrolyse aesculin, CAMP +ve, Catalase +ve, Oxidase –ve, Indole –ve. Acid
production from glucose and rhamnose, but not from xylose and mannitol. Nitrates not reduced.

RESISTANCE

0
It is killed by moist heat at 55 C for 40minutes and is readily susceptible to the lethal effects
of disinfectants. Under natural conditions, in summer they survive for 1month and in winter for 3-4
month.
 39

ANTIGENS AND TOXINS

Based on somatic and flagellar antigens, sofar 16 serovars have been identified. Of these 16
serovars, all cases of animal and human infections are caused by 3 serotypes. ½ a, ½ b and 4 b.
Numbers indicate O antigen and alphabet indicate H antigens.

TOXINS

 Act A: Act A protein is important in intracellular movement by actin polymerization and is also
thought to play a role in tropism and invasion.
 Cell wall: The lipoteichoic acid and peptidoglycan of the cell wall intract with macrophage
cells resulting in the release of proinflammatory cytokines.
 Internalins: Internalins are surface proteins responsible for adhesion and entry into target
cells.
 Listeriolysion O: It is a pore forming cholesterol dependant cytolysin. It releases
L.monocytogenes from the phagosome into the cytosol following phagosome acidification. It
also induces apoptopsis in hepatocytes.
 Phospholipase C – important in membrane lysis.
PATHOGENESIS

In both cattle and sheep, listeriosis can manifest itself in four ways;
- As a CNS infection (meningo encephalitis in adults and meningitis in the young)
- As abortion
- As a generalized septicaemia with involvement of the liver and other organs
- As mastitis in dairy cattle.

Silage is commonly implicated in outbreaks of Listeriosis in cattle and sheep. Most pathogenic
bacteria require the availability of iron in the host for metabolic activities. High iron levels in silage
that lead to elevated tissue concentrations of iron may predispose cattle and sheep fed on silage to
Listeriosis. Poor quality silage with a pH greater than 5.5 is favourable for multiplication of Listeria
monocytogenes.

Exposure to listeria occurs via oral route. Most listeria are destroyed by gastric acids. Use of
antacids and H2 blockers are considered as risk factors for infection. Intestinal translocation appears
to be a passive process that involves both intestinal epithelial cells and M cells.

After penetrate the epithelial barrier in the intestine and multiply in hepatic and spleenic
macrophages aided by the haemolysin named listeriolysin O. Then the listeria escapes from the
phagosome (with Listeriolysin O) become associated with actin filaments in the cytoplasm, and
propels itself to the cell’s plasma membrane via polar assembly for actin filaments (with Act A). In
this way, it is able to pass to neighbouring cells in plasma membrane protrusions and thus avoid host
defense mechanisms.

If the pathogen penetrates through damaged mucousal surfaces (oral, nasal or ocular) to the
CNS, via the neural sheath of trigemianl nerve or an alternate route it may penetrate through the
dental pulp (when sheep are cutting or losing teeth) to the CNS results in neural form.

SYMPTOMS

Four syndromes;
a) Subclinical: infections are the most common form of infection. Usually outbreak occurs when
fed with poor quality, high pH silage, particularly during cold weather.
b) Neonatal infections: Characterised as visceral infections with a septicemia. Often
gastroenteritis and bilateral meningitis. Deaths are frequent in neonatal animals.
c) Listerial abortion is a sporadic condition in cattle & sheep. Abotion is usually late
term – after 7 months in cattle and 12 weeks in sheep. The fetus may be macerated or
delivered weak and moribund. Retained placenta and metritis are common.
d) Neural Listeriosis: Encephalitis (circling disease):
 40

The incubation period is ranges from 14 to 40 days. The disease is more common in winter or
early spring. The clinical presentation of meningoencephalitis in adult ruminants may begin with signs
of depression and confusion. The ears droop; animal holds it head to one side.

Protrusion of the tongue and salivation are common and twitching or paralysis of the facial
and throat muscles may occur. When the animal moves, it tends to be in a single direction, giving rise
to the common name of circling disease. In the terminal stages, the animal may fall and be unable to
rise.

In poultry, there are signs of torticolis, weakness, incordination of legs and sudden death in
young birds. The disease is usually fatal in sheep, pigs and horses.

LESIONS

Microabscess in the brain stem, usually unilateral, together with perivascular cuffing is very
characteristic of listeriosis. The lesions are most common in the midbrain, pons and medulla
oblongata. In addition to this there will be generalized septicaemia, focal necrosis of the liver and
spleen will be seen.

DIAGNOSIS

Specimens to be collected

Visceral form : Material from lesions in liver, kidneys or spleen

Neural form : Spinal fluid, brain stem, and tissue from several sites in the medulla oblongata
Abortion : Placenta (cotyledon), foetal abomasal contents and /or uterine discharges.

1. Stained smears from lesions may reveal gram +ve rods (often coccobacillary)

2. Histopathological examination of brain tissue can often give a presumptive diagnosis of neural
listeriosis

3. Isolation and Identification

Inoculation of specimens on selective media includes blood agar with an antibiotic supplement or
blood agar containing 0.05% potassium tellurite (inhibitory to gram –ve). Specimens from the visceral
form of the disease or from abortion cases are inoculated directly onto the laboratory media. A cold-
enrichment procedure is necessary for brain tissue from neural listeriosis. Small pieces of spinal cord
and medulla are homogenized and a 10% suspension is made in a nutrient broth. The broth
0
suspension is placed in the refrigerator at 4 C and sub cultured on to blood agar once weekly for upto
12 weeks.

4. Inoculation in developing chicken embryos causes development of focal necrotic lesions on

the chorio allantoic membrane (CAM).

5. Anton’s test:

Inoculation of live bacterial suspension into the conjunctiva of a rabbit or guineapig only
L.monocytogenes causes a purulent keratoconjuctivitis within 24-36hrs of inoculation.

TREATMENT

Ruminants in early stages of septicaemic listeriosis respond to systemic therapy with

ampicillin or amoxicillin. Response to antibiotic therapy may be poor in neural listeriosis although
prolonged higher doses of ampicilins or amoxicilin combined with an aminoglycoside may be
effective. Ocular listeriosis requires treatment with antibiotics and corticosteroids injected
subconjuctivily.

CONTROL AND PREVENTION:

 41

Poor quality silage should be avoided. Vaccination with killed vaccines, which do not induce
effective cell-mediated immune response, is not protective because L.monocytogenes is an
intracellular pathogen. Live, attenuated vaccines, which contain serovars 1/2a, 1/2b, and 4b are
reported to reduce the prevalence of listeriosis in sheep.

ERYSIPELOTHRIX

SYSTEMATICS

Main host and diseases of Erysipelothrix rhusiopathiae

Main host (s) Disease syndrome

Pigs Swine erysipelas
* Acute septicaemic form (Pregneant sows may abort)
* Urticarial form (Diamond skin disease)
* Vegetative endocarditis and Polyarthritis (Chronic form)
Sheep * Poly arthritis in lambs
* Post-dipping lameness
* Valvular endocarditis and pneumonia
Turkeys, Geese and other birds * Acute septicaemia (Turkey erysipelas)
* Vegetative endocarditis and arthritis (Chronic form)
Human Erysipeloid (localized cellulitis)

NATURAL HABITAT

The bactrerium is widespread in nature and has been recovered from a wide variety of wild
and domestic animals including mammals, fish (both fresh and salt water), birds, reptiles and
amphibians. It is present in the soil and can survive for 20 days or longer in alkaline soil. The major
source of infection for swine and turkeys is carrier animals of the same species. It is reported that 30-
50% of pigs carry the bacterium in their tonsils, other lymphoid tissues. It is present in slurry of piglets
and can be recovered from the faeces of carrier pigs.

MORPHOLGY

Erysipelothrix rhusiopathiae (previously named Erysipelothrix insidiosa) from S (Smooth) -

form colonies and usually from acute syndromes is a gram-positive rod, the R (rough) form colonies
usually from chronic disease is a gram-positive filament. The organism is non-motile, non-spore
forming, non-acid fast, capsulated occur either in singly, in groups or in chains.

CULTURAL CHARACTERS

It is a facultative anarobe, but growth is enhanced by 10% CO2. It is able to grow in a

0 0
temperature range of 5 C to 42 C, within a pH range 6.7 to 9.2 and 8% NaCl2. Growth occurs on
nutrient agar but is improved by the addition of serum or blood. It will not grow on Mac Conkey agar.
Media contain either sodium azide (0.1%) or crystal violet (0.001%) may be used as selective media.

On blood agar, non-haemolytic pinpoint colonies (0.5 mm) appear at 24hrs incubation.
Colonial variation becomes obvious at 48hrs incubation when a zone of greenish haemolysis often
develops under and just around the colonies. The smooth form colonies are convex, circular with an
entire edge. The large rough form colonies are flatter, more opaque and have an irregular edge.

A characteristic reaction is produced when triple sugar iron agar is stab inoculated. When
0
incubated at 37 C for 24hrs. H2S is produced as a thin, black line just along the inoculation stab. The R
0
forms gives a bottlebrush or pipe cleaner type of growth in stab cultures of gelatin incubated at 21 C
for 5 days.

BIOCHEMICAL CHARACTERS
 42

The bacterium is coagulase positive, catalase negative and oxidase negative. It does not
hydrolyse aesculin or produce urease. Erysipelothrix rhusiopathiae usually ferments lactose, glucose,
levulose and dextrin. But the acid production is poor. To obtain the good result, carbohydrate tests
can be carried out in peptone water with added sterile horse serum (5-10%) with phenol red as the
indicator. Indole, Methyl red and Voges proskauer tests are negative.

RESISTANCE

Erysipelothrix rhusiopathiae is resistant to several chemicals including sodium azide, and to

drying, pickling, salting and smoking. It is capable of surviving for nearly a year in putrefying meat. But
they are susceptible to caustic soda and hypochlorites. They are readily killed in moist heat at a
0
temperature of 55 C for 10mts.

ANTIGENS AND TOXINS

Based on heat labile and heat stable antigens sofar 23 serotypes have been identified. Strains
of serotype 1 are subdivided into 1a and 1b. Serotypes 1a, 1b and 2 are most frequently involved in
disease in swine.

Cell wall:

The lipoteichoic acid and peptidoglycan of the cell wall interact with macrophage cells
resulting in the release of proinflammatory cytokines.

Neuraminidase:

It is responsible for adherence to cell surfaces. It causes cleavage of sialic acid residues on
endothelial cells leads to thrombus formation.

Hyaluronidase and coagulase:

These enzymes are also produced by some strains.

PATHOGENICITY

The carrier animals are an important source of the organism. Entry of the organism may be by
the oral, cutaneous or respiratory route. Ingestion of contaminated feed or water or contaminations of
abraded skin are the most common means of infection in swine.
Contaminate environment
Carrier animals

Organism ingested

Enter small intestine

Adhere to epithelium

Penetrate intestine

Blood stream

Localization Vascular damage

 43

Immune complex Thrombosis

Fever
Vascular damage
Arthritis
Endocarditis
Skin lesions

Erysipelothrix rhusiopathiae is able to adhere to epithelial cells, and that they invade the blood
stream and cause localization. The more virulent strains produce high levels of neuraminidase. That
cleaves sialic acid present on cell surfaces leads to vascular damage and thrombus formation.
Congestion of dermal capillaries results in diamond skin disease.

Localization of E. rhusiopathiae in joints of swine leads to fibrinous exudation and pannus

formation. Subsequent damage to the articular cartilage and prolonged retention of bacterial antigen
in the synovial tissue and chondrocytes are responsible for chronic infection.

SYMPTOMS

Erysipelas occurs in pigs of all ages, but pigs from 2 months to one one year age are highly
susceptible.

Four forms of clinical disease in swine have been described. These may occur alone or in
combination. They are;
 Acute septicaemia
 Urticarial or diamond skin lesions
 Vegetative endocarditis
 Arthritis

Swine erysipelas manifest in three forms - Acute, subacute or chronic course. The acute
disease is characterized by high fever, inappetance, depression, a rapid course of illness, and death
within 2-3 days in untreated animals. Some animals may show a stiff gait and reluctance to stand or
move, and urticrial cutaneous lesions may develop. The urticaria may be pink or purplish especially in
the abdomen, thighs, ears and tail. In severe cases the skin become necrotic and is sloughed. The
diamond shaped raised skin lesions is pathognomonic. Pregnant sows may abort. A vegetative
endocarditis is manifested by signs of cardiac insuffiency or sudden death. Subacute disease is
similar to the acute except that it is less severe and animals are likely to recover within 5-7 day.

In the chronic form arthritis is more common. The hock, stifle, elbow and carpal joints are
most likely to be affected resulting in severe lamness. The mitral valves are involved in valvular
endocarditis. Valvular vegetations are due to fibrin deposition and connective tissue proliferation.

Turkeys develop a cyanotic skin, become droopy, and may subsequently die. A swollen
cyanotic snood and diffuse reddening of the skin are pathognomonic.

DIAGNOSIS

Diamond shaped skin lesions are pathognomonic.

Specimens to be collected

It includes liver, spleen, heart valves or synovial tissues. Organisms are rarely recovered from
skin lesions or chronically affected joints.

Smear examination:
1. Gram-positive rods in acute cases and gram-positive filaments in chronic cases.
2. Based cultural characters and biochemical tests.
3. Serological tests are not applicable for diagnosis.

TREATMENT
 44

In addition to hyper immune serum, treatment with antibiotics such as penicillin and
tetracyclines are effective.

MYCOBACTERIA

Mycobacteria are slender rods of varying lengths that sometimes show branching
filamentous form resembling ‘fungal mycelium’. Hence, the name mycobacteria, meaning fungus like
bacteria.

Although cytochemically gram positiive, the Mycobacteria do not take up the dyes of the gram
stain because the cell walls are rich in lipids – Mycolic acid. Once a dye has been taken up by the cells
they are not easily decolourised, even by acid-alcohol. Mycobacteria are therefore called as acid-fast
bacilli.

The genus includes animal and human pathogens as well as saprophytic members often
 45

referred to as atypical, anonymous, opportunistic, tuberculoid and MOTT (Mycobacteria other than
typical tubercle) bacilli.

Clasification of Mycobacteria (Tubercle Bacilli)

I. Slowly growing mycobacteria

 Mycobacterium tuberculosis causes human tuberculosis in human and dogs.

 Mycobacterium bovis causes bovine tuberculosis in many animal species and also cause
tuberculosis in human
 Mycobacterium africanum causes human tuberculosis.
 The human type (Mycobacterium tuberculosis) is primarily a pathogen for man. But can
cause disease in cattle, pigs, dogs, monkeys, parrots and other species. The bovine type
(Mycobacterium bovis) is a common cause of disease in domestic animal particularly cattle,
pigs, cat, dogs and horse. The avian type (Mycobacterium avium) is primarily a pathogen for
birds. But can cause disease in cattle, sheep, goat and pigs.

II. Atypical mycobacteria

Runyon (1959) grouped the atypical mycobacteria on the basis of pigmentation, colonial
morphology and growth rate. The photochromogens will produce pigment only if exposed to light.
The scotochromogens are those that produce yellowish orange pigments in the dark. The slow
growing mycobacteria are those that require over 7 days incubation. And rapid growers are those
requiring less than 7 days.
1. Slowly growing photochromogens
Mycobacterium kansasi, Mycobacterium marinum, Mycobacterium simiae
2. Slowly growing scotochromogens
Mycobacterium gordonae (tap water scotochromogens)
3. Slowly growing non-chromogens
Mycobacterium avium (Avian tuberculosis)
Mycobacterium intracellulare
Mycobacterium paratuberculosis
(Johne’s disease – chronic hypertrophic enteritis in cattle)
Mycobacterium lepraemurium (Feline leprosy)
4. Rapid growing mycobacteria
Mycobacterium phlei (timothy grass bacillus)
Mycobacterium smegmatis

III. Non-cultivable mycobacteriae: Mycobacterium leprae

Addition to this the unspecified acid-fast bacilli such as Mycobacterium senegalense and
Mycobacterium farcinogens were isolated from Bovine farcy.

HISTORY

 The generic name mycobacterium (fungus bacterium) was proposed by Lehmann and
Neumann (1896).
 The first member of this genus to be identified was the lepra bacillus discovered by Hansen
(1868) – Hansen bacillus.
 Koch (1882) isolated the mammalian tubercle bacillus and proved its causative role in
tuberculosis by satisfying Koch’s postulates.
 The acid-fast property of Mycobacterium was discovered by Ehrlich (1882).
 Johne (1895) described Johne’s bacillus - Mycobacterium paratuberculosis.

NATURAL HABITAT

It has a worldwide distribution. The usual habitats of the great majority of the cultivable
mycobacteria are water and watery habitats, marshes, wet soil, streams, lakes, rivers. The source of
 46

the pathogenic mycobacteria is usually infected animals. Mycobacterium bovis is excreted in

respiratory discharges, faeces, milk, urine and semen. Mycobacterium avium and Mycobacterium
paratuberculosis are shed in faeces and Mycobacterium tuberculosis mainly in respiratory discharges.

The atypical mycobacteria are widespread in soil, pastures, grass and water. A few are
commensals in animals and may infect them.

MORPHOLOGY

They are acidfast organisms, which are usually straight or slightly curved rod occurring singly,
pairs or in small groups. The morphology varies from cells of species to species. Mycobacterium
tuberculosis is often arranged in serpentine cords (Smear prepared from liquid medium).
Mycobacterium kansasi is distinct banded or beaded appearance, while Mycobacterium avium is
often almost coccoid. In clinical materials M. avium subsp.paratuberculosis may appear as bundle of
faggots. They are non-motile, non-sporing and non-capsulated.

CULTURAL CHARACTERS

A comparatively slow growth rate is characteristic of the mycobacteria, with generation time
ranges from 14-20hours. Colonies appear only in about two weeks and sometimes may be delayed
0
upto 6-8 weeks. Optimum temperature is 37 C and pH is 6.4 –7.0.
Mycobacterium tuberculosis is an obligate aerobe while Mycobacterium bovis is
microaerophilic. Growth is stimulated by 5-10% CO2. Tubercle bacilli do not have exact growth
requirements. But they are highly susceptible to even traces of toxic substances like fatty acids in
culture media. The toxicity is neutralized by addition of serum, albumin or charcoal.

Several media both solid and liquid are available. The egg based Lowenstein Jensen medium
and Stone Brinks medium are most commonly used. Malchite green dye (0.025g/100ml) is commonly
used as the selective agent. Mycobacterium tuberculosis, Mycobacterium avium and many of the
atypical mycobacteria require glycerol for growth. However glycerol is inhibitory to Mycobacterium
bovis, while sodium pyruvate enhances its growth. On Lowenstein Jensen medium (i.e. glycerol
containing media), Mycobacterium tuberculosis giving the characteristic rough, tough and buff
colonies – is known as eugonic. On Lowenstein-Jenson (LJ) slants, M. tuberculosis typically has a
crumbly, "bread-crumb" appearance, whereas M. avium complex has a bright yellow, smooth,
characteristic colony (so-called "condom colony").

The growth of Mycobacterium avium in this media also described as eugonic. Mycobacterium
bovis has sparse, thin growth on glycerol containing media that is called dysgonic. Mycobacterium
bovis however grows well on pyruvate containing media without glycerol (i.e. stone brink medium)

Pigment formation is tested with young, well-developed colonies on Lowenstein Jensen

medium. The cultures are exposed to a 100-watt, clear electric bulb, at a distance of 50cm, for atleast
an hour and then incubated again in darkness for a further 1-3 days. After this treatment the
photochromogens will develop pigment. Many of the mycobacteria produce yellow/orange pigments
while Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium are non-
chromogenic.

In liquid media, the growth begins at the bottom, creeps up the sides and forms a prominent
surface pellicle (mould like pellicle) that may extends along the sides above the medium. Smears
made from liquid medium (Virulent stains) gives characteristic long serpentine cords, while avirulent
strains grow in a more dispersed fashion.

Supplementation of media with mycobactin (extracted from non-mycobactin dependant

isolates of M.avium subsp. paratuberculosis) is required for M.avium subsp. paratuberculosis.

BIOCHEMICAL CHARACTERS

They are oxidative. Atypical mycobacteria are catalse positive, while tubercle bacilli are
peroxidase positive. Niacin production and nitrate reduction is only by Mycobacterium tuberculosis.
Urease is reduced by Mycobacterium tuberculosis and Mycobacterium bovis but not by avian strain.
 47

RESISTANCE

The Mycobacteria are resistance to physical influences and will retain their viability in soil and
0
particles of dried faeces for many months. They are not specifically heat resistant; being killed at 60 C
in 15-20mts. Cultures may be killed by exposure to direct sunlight for two hours. Bacilli in sputum may
remain alive for 20-30hrs and in droplet nuclei for 8-10 days.

They are relatively resistant to disinfectants i.e. exposure to 5% phenol, 15% H2SO4, 3% nitric
acid, 5% oxalic acid and 4% NaOH. It is destroyed by tincture of iodine in 5mts and by 80% ethanol in 2
-10mts.

ANTIGENS AND TOXINS

Many antigens have been identified in mycobacteria. Group specificity is due to

polysaccharide and type specificity is due to protein antigen.

They do not produce any exotoxins. The cellwall of the mycobacterium is composed of
peptidoglycan, arabinogalactan and mycolic acid. In addition to this it contain wide range of lipids.
The outerlayer layer of the cellwall is composed of mycosides (Peptidoglycolipids or Phenolic
glycolipids).

Mycosides are responsible for the control of cellular permeability, resistance to action of
water-soluble enzymes, antibiotics and disinfectants.

Cord factor (Trehalose–6, 6’ dimycolate) and Wax D- inhibits chemotaxis, leukotoxic,

responsible for delayed hypersensitivity.

Sulfatides- sulfur containing glycolipids – promote the survival of virulent tubercle bacilli
within macrophages by inhibiting phagolysosome formation and avoiding exposure to hydrolytic
enzymes present in the lysozomes.

Virulence appears to reside in the lipids of the cell wall. Mycosides, phospholipids and
sulpholipids are protecting the tubercle bacilli against Phagocytosis.

PATHOGENEIS

Infection is usually by inhalation and ingestion. The mucociliary clearance by mucus and
epithelial cilia in the upper respiratory passages provides defense against infection. However,
microorganisms on small particles (1-4 μm in size), such as, dust and water droplets reach alveolar
spaces.

The infectious process begins with deposition of tubercle bacilli in the lung or on pharyngeal
or intestinal mucous membranes. In previously unexposed animals, local multiplication of the
mycobacteria occurs and the resistance to phagocytic killing allows continued intra cellular and extra
cellular replication. Infected host cells with mycobacteria can reach local lymphnodes and from there
may pass to the thoracic duct with general dissemination. Haematogenous spread may produce
milliary tuberculosis (in deer). This involves multifocal tubercle formation in an organ.

LESIONS

Cattle:

Catlle are usually infected with M. bovis. Infection enters usually by the respiratory tract.
Tuberculosis consists of a characteristic lesion – the tubercle. This is an avascular granuloma
composed of a caseous necrosis in a central area encircled by a zone of epitheloid cells, and a
peripheral zone of lymphocytes, granulocytes and fibroblasts. Calcification may be present in the
necrotic centers. An outer boundary of fibrous tissue is usually present between the lesions and
normal tissue.
 48

Tubercle lesions are more commonly present in the lymphnode, lungs and pleura.
Haematogenous dissemination leads to similar lesions in liver, kidney etc. Similar lesions are seen in
sheep and goats.

Horses:

Horses are infected more often with M. avium than with M. bovis. Infetion enters usually by
the alimentary tract. Primary complexes related to pharynx and intestine. Secondary lesions may be in
lung, liver, spleen and serous membranes.

Pigs:

Swine can be infected mostly by M. bovis via alimentary tract. M. avium infections are
predominat in many countries. Tubercles are most commonly seen in the skeleton especially
vertebrate and long bones are common sites. Meninges and viscera are also commonly affected.

Birds:

They are primarily susceptible to M.avium via alimentary tract. The grayish white
granulamatous lesions are found in the liver and they are also present in the intestines, spleen and
bone marrow. Mycobacterium genavense affects canaries and parrots.

DIAGNOSIS

Specimens

Specimens from live animals include aspirates from cavities, lymphnodes, biopsies,
tracheobronchial lavages and centrifuged deposit from about 50 ml of milk in the case of suspected
tuberculous mastitis. With dead animals, collect fresh and fixed (10% formalin) samples of lesions.

Diagnostic techniques

1. Based on history, signs and post mortem lesions

2. Direct microscopy

The Ziehl-Neelsen (acid-fast) stain is used to stain smears from lesions and other
specimens. Organisms are appearing as slender, often beaded, red staining rods against a blue
background. Smears stained by fluorescent dyes (auramine, acridine orange or fluorochrome) allow
the mycobacteria to be seen more easily if relatively small numbers are present.

3. Isolation

Several preliminary procedures are necessary in order to recover the comparatively slow
growing mycobacteria

a). Selective decontamination to reduce significantly the number of fast growing contaminating
bacteria.

b). Digestion or liquefaction of mucus is necessary. Mucin-trapped mycobacteria in specimens, such

as broncho-tracheal exudates, may not be available for growth in cultures.

c). If mycobacteria are present, they must be concentrated by centrifugation.

For deconatmination, the ground-up specimens must be treated with decontaminating agents,
such as 5% oxalic acid, 4% sodium hydroxide followed by neutralization of the acid or alkali or 20%
antiformin can be used and subsequently cultured in stone brink or Lowenstein Jensen media.

4. By animal inoculation
 49

Inoculation of Mycobacterium Mycobacterium Mycobacterium

suspected material tuberculosis bovis avium
Rabbits (I/V) + ++ ++
Guinea pig (S/C) ++ ++ -
Chicken (I/V) - - ++

5. Bacteriophage typing: A, B, C, I

6. Tuberculin test (Intra dermal, double intra dermal, ophthalmic tests in cattle and wattle test in case
of poultry)

7. Gamma interferon assay, Gas liquid chromatography, PCR, ELISA for detecting circulating
atnibodies, FAT, LTT assays can also be used in the diagnosis

CONTROL AND PREVENTION

Treatment and vaccination are inappropriate in control programmes for cattle. In many
countries, tuberculin testing followed by isolation and slaughter of reactors has been implemented as
the basis of national eradication schemes.

PARATUBERCULOSIS (JOHNE’S DISEASE)

Johne’s disease or paratuberculosis is caused by Mycobacterium avium subsp

paratuberculosis (also referred as Mycobacterium johnei). This disease is caused by chronic,
contagious fatal enteritis, which can affect cattle, sheep, goats, camels and wild ruminants.

Note: Mycobacterium avium subsp paratuberculosis infection in human called as Crohn’s

disease (Chronic enteritis in human).

MORPHOLOGY

Mycobacterium avium subsp paratuberculosis are acid-fast organism. They are short rods
measuring 1-2μm in width with rounded edges. It is motile and does not form spores. On artificial
media the organism tends to be shorter club form.

CULTURAL CHARACTERS

It requires mycobactin - Killed extracts of M.phelei or other killed acid-fast organism- enriched
media for growth. Slants of Herrold’s egg yolk medium with mycobactin are highly sutibale for
isolation of organism from specimens.

0
The slants are incubated at 37 C for upto 16 weeks and examined weekly for evidence of
growth. They produce minute grayish white, friable irregular colonies, less than 1mm in d.m., in 5-16
weeks. Isolates from sheep may be pigmented.

PATHOGENESIS

The organism shed in the faeces, milk and semen of infected animals. They remain viable in
the environment for upto one year under suitable conditions. Calves under one month of age are
highly susceptible and they are developed clinical disease than animals infected later in life.

Infection is acquired mainly through ingestion. The organism is an intra cellular pathogen and
cell mediated reactions are mainly responsible for the enteric lesion. Ingested mycobacteria, engulfed
by macrophages in which they survive and replicate are found initially in Peyer’s patches. As the
disease progress, an immune mediated granulomatous reaction develops, with marked lymphocyte
 50

and macrophage accumulation in the lamina propria and submucousa. The resulting enteropathy
leads to loss of plasma proteins and malabsorption of nutrients and water.

SYMPTOMS

Clinical signs developed after prolong subclinical phase of infection. Affected cattle are
usually more than 2 years of age when signs are first observed. In cattle, the disease is characterized
by diarrhoea, initially intermittent, dark and semisolid, but becoming persistent and profuse.
Progressive weight loss results without loss of appetite, emaciation and eventually death. The
mortality rate may approach 100%. Asymptomatic carrier cattle have an increase incidence of
mastitis and infertility. In sheep and goats, the disease is clinically evident only in mature animals. The
diarrhoea is less marked and may be absent.

LESIONS

Chronic catarrhal inflammation of the intestine is characteristic. In cattle, the mucousa of

affected areas of the terminal small intestine and the large intestine is usually thickened and folded
into transverse corrugation. The mesentric and ileocaecal lymphnodes are enlarged and odematous.
Thickening of the intestinal mucousa is less marked in sheep, and necrosis and caseation may be
present in the regional lymphmnodes.

DIAGNOSIS

1. Microscopical examination by staining the faecal smears with acid-fast stain.

2. Bacteriological examination

Materials decontaminated with 0.3% bezalkonium chloride and concentrated by

0
centrifugation and subsequentlu cultured in Herrold’s egg yolk medium and incubated at 37 C for upto
16 weeks.

3. Basen on PM lesions

4. Serological tests

Complement fixation test can be used, but are laborious and relatively insensitive. Agar gel
precipitation test has been used for confirming clinical infection. ELISA, using serum absorbed with a
M.Phlei, may detect subclinically infected animals.

5. Johnin test

a. Intra dermal Johnin test

Inoculate Johnin PPD into the skin of the neck region. The delayed hypersensitivity reaction is
measured after 48 hours. Peak response usually develops a month or so after infection.

b. Intra venous Johnin test

The intravenous Johnin test reaction is measured by increase in body temperature following
intra venous Johnin PPD injection.

6. DNA probes, which are highly sensitive, are being used to detect organisms in faeces.

CONTROL AND PREVENTION

Inactivated adjuvanated vaccines are available. A live vaccine consists of nonpathogenic strain of
Mycobacterium avium subsp paratuberculosis is inoculated subcutaneously into calves soon after
 51

birth and before 4 weeks of age. It reduces the incidence of Johne’s disease in the herd.

CORYNEBACTERIUM

Corynebacteria are gram positive, non-acidfast, non-motile, non-capsulated, small

pleomorphic rods. They frequently occur in rods, coccoid, club and filamentous shape. The term
coryneform which refers to the pleomorphic club shape of these gram-positive bacteria. (From
Coryne – meaning club). The major pathogen is Corynebacterium diptheria. Which causes diptheria in
children. Corynebacteria associated with animals are called diphtheroids.

SYSTEMATICS

The main diseases, hosts and natural habitats of the Corynebacteria are;

Spcies Main host (s) Diseases Natural habitat

C. pseudotuberculosis Sheep and Goats Caseous
(Corynebacterium ovis or (Non-nitrate lymphadenitis
Preisz Nocard Bacillus) reducing biotype) Skin, mucous
membrane and G.I.
Ulcerative
tract
Horses & Cattle lymphangitis
(Nitrate reducing
biotype) Pigeon fever or
Breast bone fever
Cattle Pyelonephritis and Prepuce and semen of
cystitis asymptomatic bulls,
vaginal mucous
Corynebacterium renale membrane of heathy
cows
Pigs Kidney abscess
Male sheep Balanoposthitis
(Pizzle rot)
Corynebacterium cystitidis Cattle Severe cystitis, Male genital tract
rarely
Pyelonephritis
Corynebacterium pilosum Cattle Pyelonephritis Male genital tract and
urine
Corynebacterium bovis Cattle Subclinical mastitis Udder and teat canal of
cows
Rhodococcus equi Foals Suppurative Soil and Faeces of
(Corynebacterium equi) (2-4 months) bronchonephritis foals and other
herbivores
Pigs Cervical Soil
lymphadenitis

MORPHOLOGY

They are gram-positive slender rod with a tendancy to clubbing at one or both ends; they are
non-sporing, non-motile, non-capsulated and non-acidfast.

They have granules composed of (high energy phosphate stores) – polymetaphosphate. The
granules are more strongly gram positive than the rest of the bacterial cell. Stained with Loeffler’s
 52

methylene blue, the granules take up a reddish purple color and hence they are called metachromatic
granules. They are called as volutin or Babes Ernst Granules. They are often situated at the poles of
the bacilli and are called polar bodies. Special stains, such as Albert’s, Neisser’s and Ponder’s have
been devised for demonstrating the granules clearly.
Stained smears from animal tissues often reveal groups of cells in parallel (Palisades) or
cells at sharp angles to each other (Chinese letter or Cuneiform arrangement). This is due to the
incomplete separation of the daughter cells after binary fission.

Rhodococcus equi can appear as a gram-positive coccus or a rod or club shaped form
arranged in clusters. It is capsulated and sometimes weakly acid fast.

CULTURAL CHARACTERS

Growth is scanty on ordinary media. Enrichment with blood, serum or egg is necessary for
0
good growth. The optimum temperature for growth is 37 C and optimum pH is 7.2. It is an aerobe and
facultative anaerobe. Sheep or ox blood agar is used routinely along with Macconkey agar to detect
any gram-negative contaminants that may be present.

On blood agar, Corynebacterium ovis colonies are small, white, dry and non-hamolytic at 24hr
incubation. A narrow zone of haemolysis occurs at 72 hrs incubation. After several days incubation
the colonies can reach 3mm in d.m. and appear dry, crumbly and cream in color.

Corynebacterium bovis colonies are small, white, dry and non-haemolytic. As it is a lipophilic
corynebacterium, they grow very well on media enriched with 0.5 –1% tween 80.

Rhodococcus equi colonies are small, smooth, shiny and non-haemolytic after 24hrs
incubation. But on 4-day culture, the colonies become larger, mucoid and salmon-pink in color. The
salmon-pink pigmentation is not easily seen against a red background. So, the mucoid colonies and
salmon-pink pigmentation can easily be demonstrated on nutrient agar (4-day culture). Nutrient agar
enriched with yeast extract and glucose is useful for enhancing the salmon pink pigmentation.

The renale groups are non-haemolytic. On nutrient agar, after 48 hrs incubation,
Corynebacterium renale produces dull yellow colonies. The Corynebacterium pilosum produces
distinct yellow and Corynebacterium cystitidis exhibit white colonies. On milk agar Corynebacterium
renale show casein digestion, while Corynebacterium pilosum and Corynebacterium cystitidis do not
give this reaction.

On CAMP tests, the Corynebacterium ovis, Rhodococcus equi and Corynebacterium renale
interacting with beta haemolytic of Staphylococcus aureus and gives the following results.

Organisms Staphylococcal β haemolysis

Corynebacterium ovis Inhibition

Rhodococus equi Enhancement

Corynebacterium reanale Enhancement

BIOCHEMICAL CHARACTERS

They are catalase positive, oxidase negative. Except Corynebacterium bovis others are urease
positive. The renale group is very strong urease positive (less than one hour). All diptheroids ferments
sugar except Rhodococcus equi. Corynebacterium bovis and Corynebacterium renale ferments both
glucose and maltose.

Two biotypes of Corynebacterium ovis are recognised. The ovine/caprine strains lack nitrate-
reducing capacity, while the equine/bovine strains usually reduce nitrate.

RESISTANCE
 53

0
Diptheroids are readily destroyed by heat, 60 C for one hour. They are highly susceptible to
disinfectants. It is more resistant to the action of light, dessication and freezing. Rhodococcus equi is
resistant to 2.5% oxalic acid for one hour.

ANTIGENS AND TOXINS

The diphtheroids antigen and toxins are not well documented. Corynebacterium ovis
produces a filterable toxin similar to that produced by Corynebacterium diptheria. It is a haemolytic
toxin, which has phospholipase D activity. The lipoteichoic acids and peptidoglycan of the gram
positive cell wall interact with macrophage cells resulting in the release of proinflammatory cytokines.
The high concentration of lipids contributes to intraphagocytic survival and leukotoxicity.

In Corynebacterium renale, the pili is antigenic, it express fibrillar protein adhesin on their
surfaces. Renalin- a Corynebacterium renale extracellular protein may play a role in lysis of cell
membranes. The lipoteichoic acids and peptidoglycan of the gram positive cell wall interact with
macrophage cells resulting in the release of proinflammatory cytokines. Urease – plays major role in
pathogenesis.

Rhodococcus equi produces diffusible Rhodococcus equi factors (Phospholipase C and

Cholestrol oxidase) and these as well as the capsule and cell wall constituents probably play a major
role.

PATHOGENESIS

The diphtheroids infections are characterized by the development of suppurative lesions and
clinical manifestations do not develop in the absence of predisposing factors.

Corynebacterium ovis

The prevalence of caseous lymphadenitis may be as high as 50% in adult sheep. Some
clinically normal sheep may carry the organism in the digestive tract, excrete in the faeces and
contaminate the environment. When bacteria enter the host via skin wounds (or tick bite), multiply and
are phagocytosed. Phagosome-lysosome fusion takes place. But Corynebacterium ovis multiplies in
the phagolysosome and phagocytic cells die. Permeability of local blood vessels increases,
encouraging the spread of infection from the initial site to other locations, often-regional lymphnodes
and produces toxin – Phopholipase-D. Abscesses may develop at either primary or secondary sites,
eventually rupturing and discharging thick, caseous pus containing large numbers of viable bacteria.
In some instances, lesions become metastatic and, as they increase in number, the thin ewe
syndrome develops, resulting in progreesive debilitation and death.

Corynebacterium renale

Corynebacterium renale is a normal flora in the lower urogenital tract. This group possess
fimbriae which allow attachment to the urogenital mucousa.The major predisposing factors that put a
cattle at risk are the shortness of the female urethera and the effects of preganancy and parturition,
thus, disease occurs most frequently in mature cows. The vulva may be an important portal entry for
Corynebacterium renale into the urinary tract. Bacteria grow readily in urine and ascend (through
vesiculouretharal reflex) to the kidney. Corynebacterium renale has high urease activity. The urease is
nephrotoxic and produces pyelonephritis.

Rhodococcus equi

Rhodococcus equi may be a commensal in the intestine of horses and it is largely a soil
organism. The soil enriched with equine faeces and summer temperatures are favours the rapid
multiplication of this bacterium. The disease is usually seen in 2-4 month old foals, possibly due to
the decline in maternal antibody at about 6 weeks of age. The main route of infection is by inhalation.
Rhodococcus equi is a facultative intracellular pathogen. Its ability to survive, persist in and eventually
to destroy alveolar macrophages is the basis of its pathogencity. It causes granulomatous
inflammation and abscesses in the lung tissue. Heavily infected sputum may be swallowed by the
 54

affected foal leading to ulcerative colitis and mesentric lymphadenitis.

SYMPTOMS

Corynebacterium ovis

Caseous lymphadenitis in sheep is characterized by skin wounds, enlarged lymph glands and
abscess distributed throughout the body. But the disease can be a mild infection and it is unnoticed
until postmortem is done.

Ulcerative lymphangitis in horses are similar to glanders. Initially there is a pain and swelling
of the hind limbs and enlargement of lymphatic vessels. Ulcers develop, starting at the fetlock,
discharging purulent material. Its progress towards the inguinal region is marked by swelling and
abscesses. In severe cases it spreads to abdomen, forelegs and neck leads to death.

Pectoral abscess is also called pigeon fever and breast bone fever. C. ovis causes abscesses,
usually in the muscles of the chest and caudal abdominal region of horses. Signs such as swelling,
pain and lameness depend on the location and size of the abscess may be observed. Septicaemia
may result in renal abscess, debilitation and death.

Corynebacterium renale

In pyelonephritis, characteristically frequent passage of turbid or blood stained urine by

animals, which are pregnant or recently calved. Restlessness and kicking at the abdomen may
indicate renal pain. The urine contains red blood cells, pus cells and albumin.

Ovine posthitis or Ulcerative balanoposthitis (Pizzle rot), particularly in common in Merino

sheep and Angora goats, is characterized by necrotizing inflammation and ulceration around the
preputial orifice, with a brownish crust developing over the lesion. Disease develops in the presence
of the urealytic agent in an area constantly irrigated with urine. Pizzle rot typically occurs in animals
on rich legume pasture that is high in proteins, which increase urea excretion, and estrogens, which
cause preputial swelling and urine retention in the sheath.

Rhodococcus equi

In suppurative bronchopneumonia, the foals develop cough, fever and increased respiratory
rate. In severe cases purulent discharge from nose just before death.

LESIONS

Corynebacterium ovis

In caseous lymphadenitis the superficial lymphnode contains a mass of greenish yellow

caseation in concentric layers, which have an onion ring appearnce in, cross section (hence, named it
as Corynebacterium pseudotuberculosis). In advanced cases, similar lesions are seen in lungs, kidney,
liver and spleen.

Corynebacterium renale

The kidneys are enlarged, necrosis and suppuration in the medulla. Wedge shaped
suppurative foci in the cortex. The kidney pelvis contains blood and pus. The bladder contains blood
and pus with petechial haemorrhages and ulceration of the tract.

Rhodococcus equi

Characterized by pyogranulamatous lesions with abscesses in the lung, associated

lymphnodes and pus in the bronchi are very characteristic.

DIAGNOSIS
 55

Specimens

Pus or exudates are collected from suppurative conditions and mid stream urine for isolation
of the Corynebacterium renale. A tracheal wash technique with infusion of saline can be used for the
recovery of Rhodococcus equi from affected foals. Diagnosis is mainly based on history, symptoms
and lesions.

Isolation and identification of the organism

Based on microscopical appearance, colonial morphology on blood agar, CAMP tests and
Biochemical tets are used for identification of the organism.

Control and Prevention

Diphtheroids are susceptible to penicillin, tetracycline, erythromycin, lincomycin, neomycin

and gentamicin.

ACTINOMYCETES

The actinomycetes comprise a heterologous group of prokaryotes that have the ability to
form gram positive, branching filaments of less than 1μm in d.m. The main animal pathogens in the
actinomycetes are in the genera Actinomyces, Arcanobacterium, Actinobaculum, Nocardia and
Dermatophilus. Non-pathogenic, prolific producers of antimicrobial substances – streptomyces are
also included in Actinomycetes.

ACTINOMYCES

NATURAL HABITAT

The actinomyces species are present on mucous membrane of the host animal, often in the
oral cavity, tonsils, and nasopharynx. The soil is the natural habitat of many actinomyces species.

HISTORY
 56

The generic name Actinomyces was first used by Harz (1879). Boestrom (1891) isolated
Actinomyces bovis. Cummins (1962) clearly demonstrated Actinomyces were bacteria and they are
distinct from other branching genera.

Diseases caused by the pathogenic actinomycetes;

Actinomycete Host (s) Disease

Actinomyces bovis Cattle Bovine actinomycosis (Lumpy jaw)
(Syn: Ray fungus) Horses Poll evil and Fistulous withers (occur as a mixed
infection with Brucella species)
Arcanobacterium pyogenes Cattle, Sheep Chronic or acute suppurative mastitis, suppurative
(Actinomyces pyogenes) and Pigs mainly pneumonia, septic arthritis, vegetative endocarditis
(Cattle), endometritis, umbilical infections, wound
infections and Seminal vesiculitis (Bulls and
Boars). Summer mastitis – a mixed infection with
Peptostreptococcus indolicus
Actinomyces viscous Dogs Canine actinomycosis
1. Localised cutaneous granulamatous abscess
and/or
2. Pyothorax and granulomas in the thoracic cavity
Actinomyces isralii Human Human actinomycosis
Actinobaculum suis Pigs Pyogranulamatous mastitis, ascending
(Actinomyces suis) pyelonephritis, cystitis.

MORPHOLOGY

The organisms show considerable pleomorphism. Actinomyces species are usually long and
fialmentousalthough short V, Y, and T configuration also occur. In lesions of actinomycosis, the pus
contains small pale yellow granules referred as sulfur granules. The sulphur granule is composed of
bacterial filaments and mineralized calcium phosphate of host origin. When the granules are crushed
and gram stained, a mass of gram-positive branching filaments about 1μm in width, short rods, and
cocci are evident. Around this mass, a circle of club shaped bodies with their narrow ends pointing
towards the centre-staining gram negative. Hence, called ray fungus. They are non-acid fast, non-
spore forming, nonmotile, non-capsulated and do not form endospores or conidia.

In case of Arcanobacterium pyogenes infections the pus or mastitic milk does not contain
any granules. Gram stained smears reveal large numbers of small, highly pleomorphic, gram-positive
rods, cocci and pear shaped cells. Occasionally short branching typical Chinese letter appearances
are also seen.

CULTURAL CHARACTERS

They cannot grow on Sabouraud dextrose agar. Actinomyces require enriched media for
growth. They grow well on sheep or ox blood agar. Actinomyces bovis is a capnophilic (i.e. required 5-
10% CO2 for its growth). Arcanobacterium pyogenes and Actinomyces viscous will grow aerobically
but 5-10% CO2 will enhance their growth. Actinomyces bovis and Actinomyces viscous usually require
2-4 days but the growth of Arcanobacterium pyogenes can usually be seen in 24 hrs.

Actinomyces bovis colonies are non-haemolytic, very small (< 1nm), white, rough or smooth
and adhere tenaciously to solid medium. Gram stained smears show gram positive, slightly branched
filaments or short forms. On subculture, the bacterium may become diphtheroidal or coccobacillary.
Actinomyces bovis grows well in thioglycollate medium giving a characteristic diffuse growth in about
7-10 days. In broth cultures, it grows in coarse aggregates, which in some cases may result in a
granular deposit with a completely clear supernate.

Arcanobacterium pyogenes produce a hazy- haemolysis after 24hrs incubation along the
streak lines. At 48 hrs incubation, the colonies are surrounded by a narrow zone of complete
haemolysis. Arcanobacterium pyogenes has the ability to pit a loeffler serum slope in 24-48 hrs. (i.e. A
loopful of pureculture of the medium is taken and a heavy inoculum is made in a small area in the
center of the slope, taking care not to break the surface of the medium. The medium is incubated at
 57
0
37 C for 24 –48 hrs).

Arcanobacterium pyogenes will give positive CAMP test with Staphylococcus aureus (i.e.
enhancement of staphylococcal haemolysis). In litmus milk, the organism produces acid and clot
after 3 days growth. Actinomyces viscous commonly produces two colonial forms, one being smooth,
entire, convex and glistening and the other is smaller, rough dry and irregular. Neither is haemolytic.
The larger colonial type yields gram-positive diphtheroided forms and the smaller colony has short
branching filaments.

BIOCHEMICAL TESTS

Both Arcanobacterium pyogenes and Actinomyces bovis are catalase negative, ferments
several sugars and produce acid. Reduction of nitrate is negative. Actinomyces viscous is catalse
positive.

RESISTANCE

0
Actinomyces are killed at a moist heat temperature of 60 C for 20 mts and they are
susceptible to various disinfectants.

ANTIGENS AND TOXINS

With the exception of Arcanobacterium pyogenes, Actinomyces species have not been shown
to produce any toxin. Arcanobacterium pyogenes produces a haemolytic exotoxin (Pyolysin O),
which is dermonecrotic and lethal and it also produces a protease and an extracellular neuraminidase.

PATHOGENESIS

Actinomyces bovis, present as part of the normal flora of the mouth. Trauma to the tissues is
the initiating event in disease and may occur as a result of shedding of teeth or as a result of coarse
feed. Whenever there is a trauma, the organism invades a variety of tissues and often produces
lesions on bone. Growth of the organism may involve maxillary bone, tongue, pharynx, lungs,
lymphnodes and S/c tissues of the head and neck. It initiates rarefying osteomyelitis and soft tissue
reaction, the condition being referred to as lumpy jaw. Granulation, mononuclear infiltration and
fibrosis occur in the lesions with sinus tracts leading to the outside. Exudate from the tracts contains
pus with sulphur granules. Arcanobacterium pyogenes is a commensal on the exposed mucosal
surfaces of cattle, sheep and swine.

Arcanobacterium pyogenes infection is often a sequel to earlier tissue injury, or to infection

with other bacteria. (i.e. Fusobacterium necrophorus, Peptostreptococcus indolicus). It produces
toxins and established mastitis with abscess formation. The acute bovine mastitis is refered as
summer mastitis. Actinomyces viscous serotype 1 appears too responsible for disease in dogs. Two
syndromes can occur, either separetly or together. One is a localized granulomatous lesion involving
skin and subcutis; the other is a pyothorax, with granulomas in the thoracic cavity and often a large
accumulation of sanguinopurulent pleural fluid containg soft white granules.

SYMPTOMS

In case of lumpy jaw in cattle there is marked swelling associated with suppurative (sinus
tract containing pus) and proliferative chronic rarefying osteomyelitis in the region. There may be
dislodgement of teeth, inability to chew and mandibular fractures. Arcanobacterium pyogenes
infection occurs most frequently in heifer and dry cows during summer months. Hence, it is named as
summer mastitis. The affected quarter become enlarged and firm. It often takes a destructive course,
causing abscess formation and sloughing. Animals have fever with general toxaemia.

LESIONS

Area of suppuration accompanied by granulation tissues is seen in lumpy jaw. Rarefying

osteomyelitiss leads to replacement of normal bone by porous bone (irregularity and honey combed)
with sinus tract containing pus. The pus characteristically contains small sulphur granules. In summer
 58

mastitis, abscess develops at any site containing greenish yellow foul smelling pus.

DIAGNOSIS

1. Direct microscopy

The pus or exudate is placed in a Petridish and washed carefully with a little saline to expose
the yellowish sulphur granules of Actinomyces bovis or the softer greyish white granules of
Actinomyces viscous. If it is stained with Gram’s, the ray fungus can be demonstrated.

2. Isolation and Identification of organism

3. FAT

4. Pitting of loeffler serum slope and CAMP test in case of Arcanobacterium pyogenes

Control and Prevention

Actinomycetes are highly sensitive to tetracycline, chloramphenicol and pencillin including

benzyl pencillin and ampicillin.

NOCARDIA

Nocardia has the ability to form gram-positive, branching filaments of less than 1um in
diameter. It is closely related to Corynebacterium, Mycobacterium and Rhodococcus species

Diseases caused by the pathogenic Nocardia

Species Host (s) Disease

Nocardia asteroides Dogs / Canine nocardiosis
Cats 1. Localised cutaneous granulamatous abscesses
2. Pyothorax and granulomas in the thoracic cavity

Cattle Chronic granulamatous mastitis

Nocardia farcinica Cattle Bovine farcy in tropical regions
Nocardia nova Dogs and Mycetoma
cats

HISTORY

Nocard described this organism in 1888, following its isolation from a case of bovine 'farcy',
hence the name of the type species: Nocardia farcinica.

NATURAL HABITAT

Nocardia species are soil borne saprophytes

MORPHOLOGY

They are gram-positive, obligate aerobes ability to form branching filaments. Some produce
true mycelia and some strains are acid-fast. All species are non-motile. Gram stained smears from
lesions revealed gram-positive branching filaments that often show fragmentation into coccobacillary
elements. The modified ZN stained smears exhibit a similar morphology but most of the filaments
retain the carbol-fuchsin dye and stain red.
 59

CULTURAL CHARACTERS

0
The Nocardia species grow very well in blood agar incubated aerobically at 37 C for upto 7
days. Inoculate the suspected colonies from blood agar into Sabouraud dextrose agar (SDA) and
0
incubated at 37 C for upto 10 days.

COLONIAL MORPHOLOGY

The colonies on blood agar often a vivid white and powdery, if aerial filaments and spores are
formed. Occasionally the colonies are smooth, heaped and variably pigmented. Both types of colonies
are firmly adherent to the agar surface. The colonies on SDA are dry, wrinkled and yellow, becoming
deep orange color with age.

MICROSCOPIC APPEARANCE

Gram-stained smears from colonies show gram-positive branching filaments that

characteristically break up into rods or coccobacillary elements with age. An MZN - stained smear
from young culture reveals red staining, branching filaments.

There are three morphological forms:

a. Group I strains have limited mycelia development due to early fragmentation of hyphae into
coccoid forms within 2 to 14 hours of incubation;

b. Group II strains produce mycelia, which fragment in about 18 to 20 hours after incubation, though
these mycelia break up into mycelial fragments within two days of growth

c. The pathogenic Nocardia species belong to Group III. The colonies are usually leathery in
appearance and pigmented. Extensive mycelium produced because fragmentation does not begin
until after 5 days incubation.

BIOCHEMICAL CHARACTERS

To differentiate Nocardia species tests such as decomposition of casein, hypoxanthine,

tyrosine, urea and xanthine are useful. They are oxidative and Catalase positive. Reduced nitrates to
nitrites. Gelatin not hydrolyzed.

TOXINS

The cell wall of nocardia is typical of Gram-positive bacteria, with the addition of
arabinogalactan, mycolic acids and high concentration of lipids. No exotoxins are known. But a
superoxide dismutase acts as a virulence factor.

PATHOGENESIS

Nocardia are aerobic and essentially saprophytic. They cause suppurative and
pyogranulamatous reactions in immunocompromised hosts or animals that have been exposed to
large doses of the bacterium. The pathogenic nocardia survive within phagocytic vacuoles by
preventing phagolysosome formation.

This is probably due to the surface lipids as Nocardia species have a cell wall similar to the
mycobacteria. Other cell wall lipids may provoke the characteristic granulamatous reaction. Exudates
are sanguineopuruelnt and can sometimes contain soft granules consisting of bacteia, neutrophils
and debris. They lack the microstructure of the sulphur granules produced by some of the
Actinomyces species.

In bovine, the nocardial mastitis onset is sudden with fever, anorexia and abnormal milk
secretion. Typically infection is introduced during the dry period with intramammary mastitis therapy.
Bovine farcy is a chronic suppurative infection usually starting at the lower limbs. It involves
 60

lymphatics of the extremities or head region and the associated lymphnodes. Ulcers and discharging
sinuses form along the path of infection.

LABORATORY DIAGNOSIS

Based on direct microscopy

Soft granules are not common in exudates from N. asteroids infections. Smears made from
exudates, aspirates, granulomatous tissue and from centrifuged deposits of bovine mastitic milk are
stained by Gram and MZN stain.

Gram-stained smears revealed gram-positive branching filaments that often show some
fragmentation into coccobacillary elements. The MZN stained smears exhibit a similar morphology
but most of the filaments retain the carbol fuchsin dye and stain red.

Based on isolation and identification / Biochemical reaction

 Characteristic colonial morphology on blood and SDA agar

 Microscopic appearance and
 Biochemical reaction.

Differentiation with Actinomyces

Actinomyces infections respond well to penicillin and other commonly used antibiotics,
nocardial infections are often refractory to treatment and N. asteroides is susceptible only to limited
range of antimicrobial agentssuch as Trimethoprim-sulphamethoxazole or erythromycin.

Characters Nocardiosis Actinomycosis

Granules in exudates Not common Usually present

Filaments MZN positive + -

Fragmentation of filaments + -

Growth on SDA + -

Powdery, white colonies + -

(aerial hyphae)
Susceptibility to penicillin - +

Note:
 Infection with Nocardia is by inhalation from the environment, while Actinomyces infection
begins in the host as a normal flora invading damaged tissues.

 Disseminated disease caused by Nocardia is more common in the dogs, while granuloma
formation is the rule with Actinomyces infection and spreads by local extension.
 When there is a doubt as to whether an animal has actinomycosis or nocardiosis,
precautionary measures to preserve the anaerobic Actinomyces should be institute, including
prompt delivery to the laboratory under anaerobic condition, culturing on brain heart infusion
agar blood plates and incubating under anaerobic, microaerophilic, and aerobic conditions.
While Actinomyces fails to grow on Sabouraud agar, Nocardia grows uninhibited.
 Acid-fast staining procedure of the sample exudate before culturing will also be helpful in the
presumptive identification of the infecting agent.
 61

DERMATOPHILUS

Dermatophylaceae is a group of bacteria with mycelial filaments which divide transversely

and in at least two longitudinal planes to form masses of coccoid or cuboidal cells, which
characteristically become motile. They are gram positive, non acid fast, aerobic and produce aerial
mycelium when their growths are stimulated by 10% CO2. Dermatophilus congolensis, Dermatophilus
dermatonomus and Dermatophilus pedis are the pathogens causing variety of skin lesions in
mammals, including man. Dermatophilus congolensis causes very severe clinical disease and its
infection is most common in tropical and subtropical regions.

Diseases caused by Dermatophilus species

Dermatophilus congolensis mainly affects Cattle, horses, sheep and goats, but many animal
species and man can be infected.The disease has many names;

Cattle : Streptothricosis or Dermatophilosis

Horse : Skin Funk (Rain Rot, Rain Scald, Dew Poisoning), Grease heal
Sheep : Mycotic dermatitis (general infection), lumpy wool (wool- covered skin) and
strawberry foot rot (skin of lower leg and coronet

NATURAL HABITAT

Dermatophilus congolensis is the only species in the genus is thought to maintain itself in
small foci of infection on a carrier animal or within scab particles in dust. It can survive in scab
material for period’s upto 3 years.

HISTORY

Bovine disease was first described by Van Saceghem in 1915 in the Congo now known as
Zaire in Africa.

MORPHOLOGY

D. congolensis is gram positive, non acid fast and aerobic to facultatively anaerobic. It is
filamentous and branching. The reproductive unit of D. congolensis is the motile coccoid zoospores,
about 2 um in diameter. Upon germinating, zoospores sprout a germ tube, about 1 um thick, which
elongates and thickens, dividing both transversely and longitudinally (res ulting in a 'tram-track'-like
appearance) to form masses of coccoid or cuboidal cells, which characteristically become motile. If
the flakes of scab (collected from infections) are treated too roughly, when the smears are made, the
filaments will disintegrate and only Gram-positive cocci (zoospores) will be seen.

Developmental cycle of D. congolensis:

Free zoospore

Germ tube formation

 62

Transverse septa forming

Formation of longitudinal septa

Binary fission and cocci (0.3 to 0.4 um)

Release of mature zoospores (0.5 to 1.0um)

Transverse, horizontal and vertical septa form in the immature filaments dividing it into coccal
zoospores. When mature, these zoospores are motile by polar flagella and are infective.

CULTURAL CHARACTERS

The bac¬terium is comparatively easy to culture and grows well on sheep or ox blood agar. An
atmosphere of 5-10 per cent CO2 enhances the growth of the organism espe¬cially on primary
o
isolation. The inoculated plates are incubated at 37°C (Optimum temp. 37 C.) for up to 5 days,
although colonies may be seen after 24-48 hours incubation.

COLONIAL MORPHOLOGY

Small (about 1 mm) greyish-yellow, distinctly haemolytic colonies can be seen after 24-48
hours incubation. They are firmly adherent to the medium and are embedded in the agar. After 3-4 days,
isolated colonies can be 3 mm in diameter and are rough, wrin¬kled and a golden-yellow colour. Older colonies
can become mucoid. No growth occurs on Sabouraud dextrose agar.

MICROSCOPIC APPEARANCE

Gram-stained smears from colonies do not show the characteristic 'tram-track' appearance seen
on direct microscopy. Usually the smears reveal uniformly staining, Gram-positive, branching
filaments but sometimes coccal forms predominate.

BIOCHEMICAL CHARACTERS

D. congolensis is catalase-positive, urease-positive, gelatine - positive and produces acid from

glu¬cose, fructose and maltose. It is indole-negative, does not reduce nitrate and non-fermentative
although acid is produced from certain carbohydrates.

PATHOGENESIS

The bacterium produces disease in many animal species. It is also a zoonosis. The common
name for the disease is dermatophilosis or streptothricosis. As far as is known the bacterium is
considered an obligate parasite, living only on animals.

Infection is spread by contact, biting insects, fomites and by other unknown means. Moist
conditions and high relative humidity are known to promote the prevalence of the disease
D. congolensis causes skin infections most commonly seen in cattle, sheep, goats, horses
and polar bears in zoological collections. The infec¬tion is characterised by the formation of thick
crusts which come away easily with a tuft of hair, leaving a moist, depressed area with bleeding
points from capil¬laries.

Infections can be localised but have a tendency to spread over large areas of the body and
the morbid¬ity and mortality can be high, especially in tropical regions.

The position of the lesions varies with the predisposing conditions. In periods of high rainfall
the lesions tend to occur along the backs of animals. Where there is a heavy infestation with
Amblyomma ticks the lesions are present in the predilection sites of the ticks: dewlap, axillae, udder
and scrotum. In the dry season, in tropical regions, when feed is scarce the lesions are on the muzzle,
head and lower limbs due to the animals foraging in thorn-covered scrub.

Though the disease does not lead to death in the adult cattle, deaths in young goats and
cattle have been reported.
 63

In sheep, the disease is called mycotic dermatitis and is seen in three forms:

1) Dermatitis of the wool-covered areas of the body or lumpy wool;

2) Dermatitis of the face and scrotum; and
3) Dermatitis of the lower leg and foot, which may result in severe ulcerative dermatitis referred to as
"strawberry foot rot".

LABORATORY DIAGNOSIS

Based on Direct microscopy

Small pieces of material are shaved from the scab with a scalpel and the flakes of scab are
softened in a few drops of distilled water on a microscope slide. A smear is made, taking care to leave a
few flakes of scab material intact. The smear can be stained by either Giemsa or Gram stains.
Giemsa is the better stain to show the characteristic morphology of the bacterium. Segmenting
filaments and coccoid spores stain deep purple. The spores are seen in packets.

Based on Isolation and Identification

The organism grows well on blood agar plates within 24 to 48 hours as small grayish white
colonies turning yellow to orange upon further incubation. Although the isolation of D. congolensis
may not be necessary for a diagnosis of streptothricosis, Scab material contains many contaminants
and Haalstra's method was developed to overcome this problem.

Haalstra's Method for the Primary Isolation of Dermatophilus congolensis;

1. Grind up a small amount of scab material and place a little in 2 ml distilled water in a container and
incubated for 3.5 hours at room temperature.

2. Place the container, with lid removed, in a candle jar at room tem¬perature for 15 minutes.

3. The motile zoospores are chemotactically attracted to the carbon dioxide-enhanced atmosphere in
the candle jar and move to the surface of the distilled water. Remove a loopful of fluid from the
surface and inoculate a blood agar plate. Incubate the inoculated plate at 37°C for 72 hours under 5-
10 per cent CO2.

Identification of this bacterium does not seem to pose any problems. Lesion and typical
morphology are quite diagnostic.

Immunity

There is no known vaccine for immunization against the disease produced by Dermatophilus.
Recovery from the disease seems to confer permanent immunity to reinfection.

ESCHERICHIA COLI

INTRODUCTION

Escherichia coli (commonly abbreviated as E. coli) is a Gram-negative, facultative anaerobic,

 64

rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms
(endotherms).

Escherichia coli are common inhabitants of the terminal small intestine and large intestine of
mammals. They are often the most abundant facultative anaerobes in this environment. They can
occasionally be isolated in association with the intestinal tract of non-mammalian animals and
insects. The presence of E.coli in the environment is usually considered to reflect faecal
contamination and not the ability to replicate freely outside the intestine.

MORPHOLOGY

E.coli is Gram-negative, facultative anaerobic and non-sporulating. Cells are typically rod-
shaped, and are about 2.0 micrometers (μm) long and 0.25-1.0 μm in diameter, with a cell volume of
0.6–0.7 μm. It can live on a wide variety of substrates. Strains that possess flagella are motile. The
flagella have a peritrichous arrangement. It is motile by peritrichous flagellae, though some strains are
non-motile. Spores are not formed. Capsules and fimbriae are found in some strains.

CULTURAL CHARACTERISTICS

Escherichia coli or E.coli cells may grow on a solid or in a liquid growth medium under a
laboratory condition. Solid and liquid media may have exactly the same composition except that the
solid medium contains an extra 1.5% agar. Different E.coli clones may have different properties.
Colonies growing on solid media represent different clones. It is an aerobe and a facultative anaerobe.
Optimal growth of E. coli occurs at 37°C (98.6°F) but some laboratory strains can multiply at
temperatures of up to 49°C (120°F).

On Nutrient agar, colonies are large, thick, greyish white, moist, smooth, opaque or
translucent discs. The smooth (s) form seen in fresh isolation is easily emulsified in saline, whereas
the rough (R) form often auto agglutinates in saline. Some strains may form “mucoid”colonies. On
MacConkey agar medium, colonies are bright pink due to lactose fermentation.

On selective media (Desoxycholate citrate agar-DCA; salmonella shigella-SS medium) used

for the isolation of salmonella, their growth is inhibited; however their colonies are pink on DCA as it
contains lactose and neutral red. In broth, there is generalized turbidity and deposit which disperses
on shaking.

Temperature 37°C MacConkey Agar Eosin-methylene blue Agar

for 24 hrs
Size in mm 1 mm 1 mm
Shape Circular Circular
Colour Pink Metallic sheen
Margin Complete Complete
Elevation Slightly Raised Convex
Opacity Opaque Translucent
Consistency Soft Soft
BIOCHEMICAL REACTIONS

E. coli uses mixed-acid fermentation in anaerobic conditions, producing lactate, succinate,

ethanol, acetate and carbon dioxide. Since many pathways in mixedacid fermentation produce
hydrogen gas, these pathways require the levels of hydrogen to be low, as is the case when E. coli
lives together with hydrogen consuming organisms, such as methanogens or sulphate-reducing
bacteria.

Glucose, lactose, mannitol, maltose are fermented with acid and gas production, but sucrose
is not fermented by typical strain of E. coli. In Triple sugar iron (TSI), acid and gas are produced. The
four biochemical tests widely used for enterobacteriaceae classification are Indole (I), Methyl Red
 65

(MR), Voges Proskauer (VP) and Citrate (C) utilisation which are referred to by the mnemonic IMViC.
Escherichia coli is Indole and MR positive VP and citrate negative (IMViC ++--), H2S is not formed and
urea is not hydrolysed.

Test Reactions
Catalase +
Oxidase –
Urease –
TSI Acid butt,
with gas, acid slant
MR +
VP –
Nitrate +
Citrate –
Indole (TW) +
Gelatin –

Key: + = reaction positive

– = reaction negative

ANTIGENS AND TOXINS

Surface Antigens of E.coli

The capsular (K) antigens are polysaccharides and the cell wall or somatic (O) antigens arc
determined by the sugar side-chains on the lipopolysaccharide molecules of the outer membrane. The
flagellar (H) and fimbrial (F) antigens are proteins. Some of the well known fimbrial antigens, K88 (F4)
and K99 (F5) are adhesins that allow palhogenic E.coli strains to adhere to intestinal cells and
colonise the small intestine. The O, H and K antigens can be used to serotype strains of E.coli; each
serotype is designated by the numbers of the antigens that it bears, for example O157:K85:H19.

E. coli has Three Antigens:

O - Somatic, Greek Ohne Hauch—without flagella;

H - Flagella; Greek Hauch—flagella and

K - Kapsular antigens.

K antigen is an envelope antigen, which encloses the O antigen, renders the strain
inagglutinable by the O antiserum and contributes to virulence by inhibiting phagocytosis.

It may be of three types —L, A and B. Though L type is common, the B antigen is medically
important as it is found on enteropathogenic E. coli.

F (Fimbrial) Antigen:

The F antigen has no significance in antigenic classification of E. coli. Type I fimbriae

mediates adhesion of bacterium to human and animal cells. Such adhesion enhances bacterial
pathogenicity e.g. urinary tract infection in which type I fimbriae has some possible role to play.

Several fibrin structures resembling fimbriae have been demonstrated. They, most probably,
play a very important role in pathogenesis of diarrhoeal diseases and urinary tract infection.

Toxin:

Besides the endotoxin associated with O antigen, some strains produce two types of
exotoxin—enterotoxin and haemolysin.
 66

Enterotoxins responsible for diarrhoea are of two types—heat labile (LT); heat stable (ST). LT
is similar to cholera enterotoxin antigenically and in its mechanism of action—by stimulating the
adenyl cyclase - cyclic adenosine monophosphate (cAMP) system to produce fluid accumulation in
the intestinal lumen. ST appears to stimulate fluid secretion into the gut through the mediation of
cyclic guanosine monophosphate (cGMP) resulting into dehydration.

Haemolysin by Escherichia Coli:

Three types of haemolysins produced by E.coli are not related to pathogenesis. E. coli forms
a part of normal intestinal flora of man and animal and the commensal strains belong to several O
groups. There are many strains of E.coli which include commensal strains as well as strains with
virulence determinants that cause a wide variety of infections of all age groups of men and animals.

The virulent strains of E. coli are specific pathogens in the gut (enteritis) and of extra-
intestinal sites (urinary tract infection, wound infection).

The worst type of E.coli, known as E.coli O157:H7 causes bloody diarrhea and can sometimes
cause kidney failure and even death. E.coli O157:H7 makes a toxin called Shiga toxin and is known as
a Shiga toxin-producing E.coli (STEC).

PATHOGENESIS AND PATHOGENICITY

The virulence factors of pathogenic strains of E.coli include capsules, endotoxin, and
structures responsible for colonization, enterotoxins and other secreted substances. Capsular
polysaccharides, which are produced by some E. coli strains, interfere with the phagocytic uptake of
these organisms. Capsular material, which is weakly antigenic, also interferes with the antibacterial
effectiveness of the complement system.

Endotoxin, a lipopolysaccharide (LPS) component of the cell wall of Gram-negative organisms,

is released on death of the bacteria. It is composed of a lipid A moiety, core polysaccharide and
specific side chains. The role of LPS in disease production includes pyrogenic activity, endothelial
damage leading to disseminated intravascular coagulation, and endotoxic shock. These effects are of
greatest significance in septicaemic disease.

Fimbrial adhesins which are present on many enterotoxigenic strains of E.coli allow
attachment to mucosal surfaces in the small intestine and in the lower urinary tract.

Two types of enterotoxins, heat labile (LT) and heatstable (ST) have been identified. Each
type of enterotoxin has two subgroups. Many strains of enterotoxigenic E.coli (ETEC) from pigs
produce LT1 which induces hypersecretion of fluid into the intestine through stimulation of adenylate
cyclise activity.

Verotoxins (VT) are similar structurally, functionally and antigenically to the Shiga toxin of
Shigella dysenteriae. These toxins are heat-labile and lethal for cultured Vero cells. Verotoxigenic
E.coli (VTEC) colonizing the intestines can damage enterocytes and, when verotoxin is absorbed into
the bloodstream, it exerts a deleterious effect on endothelial cells in relatively defined anatomical
locations such as the central nervous system in pigs. The verotoxin VT2e is implicated in oedema
disease of pigs.

CLINICAL INFECTIONS

Clinical infections in young animals may be limited to the intestines (enteric colibacillosis,
neonatal diarrhoea), or may manifest as septicaemia (colisepticaemia, systemic colibacillosis) or
toxaemia (colibacillary toxaemia). In older pigs, post-weaning enteritis and oedema disease are
manifestations of toxaemia. Non-enteric localized infections in adult animals, many due to
 67

opportunistic invasion, can involve the urinary tract, mammary glands and uterus.

In poultry, airsacculitis and pericarditis may develop following septicaemia. Coligranuloma

(Hjarre's disease) is characterized by chronic inflammatory changes which are encountered at
postmortem in laying hens and resemble tuberculous lesions.

Infection of the mammary glands of cows and sows by members of the Enterobacteriaceae,
including E.coli occurs opportunistically. In dairy cows, the source of infection is faecal contamination
of the skin of the mammary gland and relaxation of the teat sphincter following milking increases
vulnerability to infection.

Cows with low somatic cell counts are particularly susceptible to infection. No specific
serotypes of E.coli have been linked with this form of mastitis. The acute form of the disease is
characterized by endotoxaemia and can be life-threatening. Peracute disease may be fatal in 24 to 48
hours. Affected animals are severely depressed with drooping ears and sunken eyes. Mammary
secretions are watery and contain white flakes.

DIAGNOSTIC PROCEDURES

The age and species of the affected animal, the clinical signs and the duration of illness may
suggest the type of infection and the category of disease. The history, progress of the disease and the
system or organ affected influence the selection of specimens, the laboratory procedures for
diagnosis and appropriate treatment and control measures.

Suitable specimens include faecal samples from animals with cnteric disease, tissue
specimens from cases of septicaemia, mastitic milk, samples of mid-stream urine and cervical swabs
from suspected cases of pyometra or metritis.

o
Specimens cultured on blood and MacConkey agar are incubated aerobically at 37 – 42 C for
24 to 48 hours.

Identification criteria for isolates:

 On blood agar the colonics are greyish, round and shiny with a characterisitic smell. Colonies
may be haemolytic or non-haemolytic.

 On MacConkey agar colonies are bright pink.

 IMViC tests can be used for confirmation

 The colonies of some E.coii strains have a metallic sheen on EMB agar.

 A full biochemical profile may be necessary to identify isolates from coliform mastitis or
cystitis.

 Some serotypes are found in association with certain disease conditions. Slide agglutination
tests for ‘O’ and ‘H’ antigens are employed for serotype identification.

Enterotoxins in the small intestine can be detected, using methods employing monoclonal
antibodies. Some of thesc reagents are available commercially. For expression of fimbrial antigens,
isolates should be subcultured on Minca medium. Fimbrial antigens can be identified using ELISA or
latex agglutination. DNA probes specific for genes encoding heat-labile and heat-stablc enterotoxins
may be used to identify cnterotoxigenic strains of E.coli.

TREATMENT
 68

Milk feeding can be resumed gradually when clinical improvement is evident. Severely
dehydrated calves require parenteral fluid replacement therapy. Calves with
hypogammaglobulinaemia can be given bovine gammaglobulin intravenously. In most domestic
species, enteric diseases may be treated by oral administration of antimicrobial compounds which
are active in the gastrointestinal tract. Systemic and localized infections require parenteral
administration of therapeutic agents. Treatment should be based on susceptibility testing of isolates.

CONTROL

Newborn animals should receive ample amounts of colostrum shortly after birth. Colostral
antibodies can prevent colonization of the intestine by pathogenic E.coli. Absorption of
gammaglobulin from the intestine declines progressively after birth and is negligible by 36 hours.

Vaccination is of value for a limited number of the diseases caused by E.coli. Commercially
available killed vaccines containing prevalent pathogenic E.coli serotypes can be given to pregnant
sows. Alternatively, autogenous, killed vaccines prepared from strains of E.coli implicated in disease
outbreaks on a farm can be used. Vaccination of pregnant cows with purified E.coli K99 fimbrial or
whole-cell preparations, often combined with rotavirus antigen, can be used to enhance colostral
protection.

DISEASES CAUSED BY ESCHERICHIA COLI

Animals involved Disease Clinical signs and pathogenesis

PIGS

Piglets less than 1 Neonatal diarrhoea Profuse watery diarrhoea and severe dehydration,
week old (colibacillosis) mortality 90-100%

Colisepticaemia
Occasionally septicaemia with invasive strains and
death of a piglet within 48 hours of birth
Piglet meningitis
Acute meningitis and fibrinous polyserositis in
piglets has been reported
Weanling enteritis
Pigs about 2 weeks (colibacillosis) Diarrhoea, anorexia and fever. Mortality lower than in
after weaning neonatal pigs
Enterotoxins involved (STb and LT)
Oedema disease
Weaned pigs Often sudden death. Oedema of forehead, eyelids,
stomach wall and larynx (hoarse squeal). Nervous
signs such as ataxia, convulsions and paralysis may
be seen.
Verotoxin (variant of SLT-2 toxin) involved often
associated with E. coli 0139 and 0141
Coliform mastitis
Sows after farrowing One or more mammary glands affected

Gilts after farrowing Mastitis- metritis-

agalactia (MMA) Complex syndrome involving hysteria, hormonal
syndrome imbalance and coliform infection (often E. coli)

CATTLE
 69

Calves less than 'White scours' The appetite is normal at first but decreases as the
1-week old (coli bacillosis) faeces become more fluid. White pasty faeces
around rectum.
Dehydration and emaciation occur. Death usually
within 4- 5 days if untreated. Enterotoxins involved
(Sta) and possibly a Shiga-like toxin in some cases.

Sudden death due to endotoxic shock or diarrhoea,

Calves less than Colisepticaemia depression, respiratory distress and death.
1-week old Endotoxin mainly involved from invasive E. coli
strains

E. coli localised in joints and/or kidneys ('white

Calves surviving a Joint ill spot'). Entry can be via the umbilicus
Colisepticaemia
Peracute disease: fever, anorexia, depression and
Dairy cows soon after Coliform mastitis sunken eyes. Death due to endotoxic shock. Most
parturition common in housed cows

SHEEP

Neonatal lambs Colibacillosis and Syndromes similar to those that occur in calves but
Colisepticaemia less common. Enterotoxigenic and enteropathogenic
strains involved in colibacillosis and E. coli 078 is
common in
Colisepticaemia

Neonatal lambs 'Watery mouth' The lamb is dull and anorectic, saliva drools over the
muzzle and there is abdominal tympany. There are
splashing sounds within the abomasum and death
within 6 - 24 hours.

Associated with E. coli endotoxaemia

Ewes Coliform mastitis Peracute and similar to the condition in cows. Most
commonly seen in ewes housed during lambing

DOGS (CATS)

Neonatal pups Colisepticaemia Septicaemia, often fatal, associated with the

progesterone-stimulated endometrium.

Bitches Pyometra A vaginal discharge may occur 4-8 weeks after

oestrus. In a closed-cervix pyometra the bitch is
toxaemic and ill. Endotoxin involved

Adult dogs Urinary tract Cystitis (often in bitches) is most common but E.
infection coli can ascend higher in the urinary tract. A
5
bacteriuria occurs with greater than 10 E. coli /ml
urine

POULTRY

Young chicks Omphalitis Infection of the vestigial yolk sac. The contents are
dark, fluid and evil-smelling. Common name is
'mushy-yolk disease'

All ages Colisepticaemia Primary or secondary infection via the intestines or

respiratory tract. Many body organs are affected
with airsacculitis,
peritonitis and ovarian infection
 70

Chronic condition, possibly following a

All ages Coligranuloma colisepticaemia. There are nodular lesions in the
liver and intestines
OTHER ANIMALS

Neonatal animals Colibacillosis and Diarrhoea and septicaemia. Less common than in
such as foals and colisepticaemia calves and piglets
rabbits

KLEBSIELLA

Klebsiella pneumoniae (also known as Friedlander's bacillus) is a Gram-negative, non-motile,

encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal
flora of the mouth, skin, and intestines.

Klebsiella spp. are Gram-negative, nonmotile, usually encapsulated rod-shaped bacteria,

belonging to the family Enterobacteriaceae. These bacteria produce lysine decarboxylase but not
ornithine decarboxylase and are generally positive in the Voges-Proskauer test. Members of the
Enterobacteriaceae family are generally facultative anaerobic, and range from 0.3 to 1.0 μm in width
and 0.6 to 6.0 μm in length. Klebsiella spp. often occurs in mucoid colonies. The genus consists of 77
capsular antigens (K antigens), leading to different serogroups.

Morphology of Klebsiella pneumoniae (K. pneumoniae)

 Shape – Klebsiella pneumoniae is a short, plump, straight rod shape (bacillus) bacterium.

 Size – The size of Klebsiella pneumoniae is about 1–2 µm × 0.5–0.8 µm (micrometer).

 Arrangement Of Cells – K. pneumoniae is arranged singly, in pairs, or in short chains and

sometimes in clusters.

 Motility – Klebsiella pneumoniae is a non-motile bacterium.

 Flagella – K. pneumoniae is a non-flagellated bacterium.

 Spores – The Klebsiella pneumoniae is a non–sporing bacterium.

 Capsule – Capsules are present in Klebsiella pneumoniae which can easily be demonstrated
using India ink preparation, appear as a clear halo in a dark background.

 Gram Staining Reaction – Klebsiella pneumoniae is a Gram -ve (Negative) bacterium.

Culture requirements of Klebsiella pneumoniae (K. pneumoniae)

Special requirements – Klebsiella pneumoniae have no complex nutritional requirements and

readily grow in an ordinary media like Nutrient Agar medium (NAM). Commonly the NAM
& MacConkey Agar medium is used for the cultivation of Klebsiella pneumoniae in Laboratory.

There are various culture media used for the cultivation of Klebsiella pneumoniae in the
 71

laboratory and most commonly the Nutrient Agar Medium and MacConkey Agar Medium is used, the
other media are as follows;

 Columbia Horse Blood Agar medium

 Sheep Blood Agar medium

 Trypticase Soy Agar medium

 Eosin Methylene Blue Agar (EMB Agar) Medium.

 The liquid medium (Nutrient Broth medium, TSB medium)

 The Eosin Methylene Blue Agar (EMB Agar) medium which is the Selective medium
for Klebsiella pneumonia.

In Liquid culture media like Trypticase soy broth or Nutrient broth, the growth of the bacterium
occurs as turbidity in the broth medium which is further analyzed for the morphology (under the
microscope), gram reaction, biochemical tests, and Klebsiella pneumoniae specific tests.

In Blood Agar medium, the Klebsiella pneumoniae colonies are non-hemolytic i.e. shows
Gamma Hemolysis (γ-hemolysis).

In MacConkey Agar medium, the colonies of Klebsiella pneumoniae are pink colored due to
the lactose fermentation which is of great importance in differentiating K. pneumoniae from other
Bacteria present in the specimen, especially from Gram-positive bacteria and Salmonella species
which are non–lactose fermentors and gives colorless colonies on MacConkey agar medium.

In Eosin Methylene Blue Agar (EMB) medium, the colonies of Klebsiella pneumoniae are Pink
to purple in color without green metallic sheen which is of great importance in differentiating K.
pneumoniae from other bacteria normally found in Specimen and especially from E. coli which grows
with Green metallic sheen.

Key biochemical reactions:

Oxidase - negative
Catalase - positive
Indole - negative
DNase - negative
Voges-Proskauer - positive
Urease - positive
H 2S - negative
Lysine-decarboxylase - positive

HOST RANGE

Humans, mammals (including horses, bovines, rhesus and squirrel monkeys, guinea pigs,
muskrats, lemurs, and bats), aquatic animals (including elephant seals, California sea lions, and
harbor seals), reptiles (including snakes, crocodiles, and American alligators), birds, insects, and
plants (banana, rice sugar cane and maize).

SUSCEPTIBILITY TO DISINFECTANTS
 72

Gram-negative bacteria are generally susceptible to a number of disinfectants, including

phenolic compounds, hypochlorites (1% sodium hypochlorite), alcohols (70% ethanol), formaldehyde
(18.5 g/L; 5% formalin in water), glutaraldehyde, and iodines (0.075 g/L).

PHYSICAL INACTIVATION

Reduction in the growth and metabolic activity of K. pneumoniae at temperatures >35 °C has
been reported. Significant growth reduction has been demonstrated at 60 °C; however, the bacteria
still show some metabolic activity (i.e. not completely inactivated). Bacteria are also sensitive to
moist heat and dry heat.

PATHOGENICITY/TOXICITY

Pathogenicity factors of Klebsiella spp. include adhesins, siderophores, capsular

polysaccharides (CPLs), cell surface lipopolysaccharides (LPSs), and toxins, each of which plays a
specific role in the pathogenesis of these species.

Depending on the type of infection and the mode of infectivity, cells of Klebsiella spp. may
adhere and attack upper respiratory tract epithelial cells, cells in gastrointestinal tract, endothelial
cells, or uroepithelial cells, followed by colonization of mucosal membranes. Common underlying
conditions include chronic liver disease (cirrhosis), chronic renal failure, cancer, transplants, burns,
and/or use of catheters.

Respiratory disease:

 K. pneumoniae – a leading cause of community-acquired and nosocomial pneumonia and

lung abscesses. Infection of the upper lobe is more common. Symptoms include: fevers,
chills, and leukocytosis with red currant jelly-like sputum. Rare complications include lung
infection involving necrosis and sloughing of the entire lobe.

 K. ozaenae – causes ozena, a primary atrophic rhinitis (AR) which involves chronic
inflammation of the nose.

 K. rhinoscleromatis – causes rhinoscleroma (RS), a chronic granulomatous infection which

predominantly affects the cavity of the nose.

Central nervous system (CNS) infections:

 K. pneumoniae and K. oxytoca – cause community-acquired meningitis and brain abscesses.

Clinical symptoms include: fever, altered conciousness, seizures, and septic shock.

 K. ozaenae – associated with rare cases of cerebral abscess and meningitis.

Urinary tract infections (UTIs):

 Klebsiella spp. are a frequent cause of UTIs. Significant bacteriuria has been ascribed to K.
ozaenae.

Hepatic disease:

K. pneumoniae – an important causative pathogen for pyogenic liver abscesses with

symptoms including fever, right-upper-quadrant pain, nausea, vomiting, diarrhea or abdominal pain,
 73

and leukocytosis. Abscesses occur predominantly in the right lobe and are solitary.

Other infections:

K. granulomatis – causes donovanosis or granuloma, a chronic ulcerative disease that

primarily affects the genitalia. Symptoms include development of small papule or ulcer at the site of
inoculation that later develop into large red ulcers (lesions) that extend along the moist folds of the
genitalia.

PERACUTE BOVINE MASTITIS

Klebsiella species are Gram-negative coliform bacteria that can cause mastitis, leading to
significant economic losses on dairy farms. K. oxytoca and K. pneumoniae are the species that are
responsible for causing mastitis.

Source and Transmission

Like other coliforms, Klebsiella are found in manure, and manure easily contaminates the
environment of the dairy cow. Drinking water, feed, other cows, and bedding (especially wood by-
products) are some sources of environmental Klebsiella. Infection through the teats can lead to
spread of the pathogen during milking as milk from an infected cow contaminates the milking unit
and transmits the infection to the next cow that is milked.

Pathogenesis

Commonly, these organisms are found in organic matter, including bedding and manure.
Klebsiella spp. is naturally abundant on the forest floor; thus, their presence in sawdust is common.
Therefore, cows bedded on fresh sawdust are at an increased risk for mastitis caused by Klebsiella
spp.

Loads of sawdust containing high counts of Klebsiella spp. have been linked to herd
outbreaks of Klebsiella mastitis. Poor udder cleanliness, inadequate stall management, and damaged
teat ends are risk factors for Klebsiella infections in uninfected cows.

Symptoms

Of all Klebsiella cases, approximately a third are mild (abnormal milk), a third are moderate
(abnormal milk and swollen udders), and a third are severe (systemic signs, including fever, off feed,
decreased milk production, shock, or recumbency). Klebsiella invades deep into the secretory tissue
of the udder, compromising the secretory capacity of the mammary gland. Consequently, some
Klebsiella infections become chronic, and infected cows suffer a long-term reduction in milk
production.

Diagnosis

Laboratory Identification of Klebsiella:

K. pneumonia colonial morphology on blood agar is mucoid and 3 to 4mm in diameter. On

MAC, K. pneumoniae colonies are pink (LF), mucoid (usually), and 3 to 4 mm in diameter. Colonies on
Hektoen enteric agar and XLD are yellow.

(Large, mucoid, glistening pink colonies on a MacConkey agar plate are of typical colonies produced
 74

by many Klebsiella and Enterobacter spp.).

Laboratory tests:

Many commercial mini systems are available for identifying Klebsiella spp. and other
members of the family Enterobacteriaceae. K. pneumoniae is positive for Glucose, Lactose, Sucrose,
ONPG, Methyl red, Citrate, Urease, Malonate and Lysine decarboxylase. Serology Serological
identification of Klebsiella species is based on their O (body or somatic) antigens and K (capsular)
antigens reacting with pooled antisera. Klebsiella are differentiated into 72 serotypes based on the
identification of the capsular (k) antigens. The capsular identification is performed by microscopical
demonstration of capsule ‘swelling’ (Quellung reaction) in wet films with the type specific capsular
antiserum.

Treatment

Once a cow is tested positive for Klebsiella, the somatic cell count (SCC) history of the cow
should be reviewed to decide whether a treatment may be needed. One or more months of a SCC
exceeding 2,00,000 cell/mL is an indication of a chronic infection and antimicrobial treatment would
be warranted.

Prevention and control

Identification of chronically infected cows is crucial in the control of transmission; an

effective on-farm culturing program should be implemented for the early detection of infected cows.
Cows with chronic mastitis should be segregated and milked last, and culled when possible. Proper
milking practices, including pre- and post-milking teat disinfection, are important for good udder
hygiene and minimizing spread of the infection during milking.

The use of a coliform mastitis vaccine (J5 bacterin) has been shown to reduce the severity of
clinical Gram-negative mastitis, which includes mastitis caused by Klebsiella spp. It is important to
remember, however, that these vaccines do not reduce the incidence of mastitis.
 75

SALMONELLA

Salmonella consists of bacilli leading to Enteric fever, Gastroenteritis, Speticaemia etc. The
important member of the genus is Salmonella typhi, which causes Typhoid fever. Salmonella are of
two groups;

(i) Enteric fever group consisting of typhoid & Paratyphoid bacilli exclusively or primary human
parasites

(ii) Food poisoning group, which are animal parasite but may infect humans causing gastrointestinal
infections

Salmonella is oxidase negative, catalase positive, indole and Voges Proskauer (VP) negative,
methyl red and Simmons citrate positive, H2S producing and urea negative.

CHARACTERISTICS

Cells are rod-shaped, non-spore-forming, and predominantly motile by means of peritrichous

flagella with diameters of around 0.7-1.5μm and lengths of 2-5μm with a few exceptions. On blood
agar, colonies are 2-3mm in diameter. Colonies are generally lactose non-fermenters. They obtain
their energy from oxidation and reduction reactions using organic sources, and are facultative
anaerobes. They produce acid from glucose usually with the production of gas, and are oxidase
negative. Most produce hydrogen sulphide except Salmonella paratyphi A and Salmonella typhi, which
is a weak producer. They are identified with a combination of serological and biochemical tests.

Salmonella species are classified and identified into serotypes according to the White-
Kauffmann-Le Minor scheme; there are more than 2,500 Salmonella serotypes that have been
described and reported.

ISOLATION OF SALMONELLA

Some farm animals are infected with Salmonella without showing signs of the illness, i.e. they
are subclinically infected. Faeces from these herds may contain Salmonella in low numbers. In food,
Salmonella may also be present in low numbers in addition to a lot of other micro-organisms, and
they may be injured. To diminish the risk of obtaining false negative results, a non-selective pre-
enrichment of faeces or food sample, a combination of two selective enrichments and plating on two
selective media are performed:

• Pre-enrichment in non-selective medium (buffered peptone water).

• Selective enrichment in Tetrathionate broth (Müller-Kauffmann) and Rappaport Vassiliadis soy

peptone (RVS) broth.

• Subcultivation on Xylose Lysine Desoxycholate (XLD) agar and on Brilliant Green agar (BGA) (or
another selective agar media).

Primary isolation media:

 Blood agar incubated in 5-10% CO2 at 35–37°C for 18-24hr.

 76

 Xylose-lysine-desoxycholate agar (XLD) agar incubated in air at 35–37°C for 18-24hr.

 Desoxycholate citrate (DCA) agar incubated in air at 35–37°C for 18-24hr.

 Brilliant Green agar (BGA) incubated in air at 35–37°C for 18-24hr.

CULTURAL CHARACTERISTICS

Salmonellae are aerobic and facultatively anaerobic bacteria growing readily on simple media
over a range of pH 6-8 & temperature 15-41°C with optimum temperature of 37°C. Colonies are large,
circular and smooth on MacConkey and Deoxycholate citrate media, colonies are colourless due to
absence of lactose fermentation.

Colonial appearance:

 Blood agar - Colonies are moist and 2-3mm in diameter.

 CLED agar - Salmonella species are non-lactose fermenters (some serotypes eg Salmonella
Arizonae and Salmonella Indiana may ferment lactose).

 XLD agar – Colonies are red, and usually with a black centre (some serotypes eg Salmonella
paratyphi A and Salmonella typhi may not produce a black centre).

 DCA agar - Colonies are colourless, and usually with a black centre (some serotypes eg
Salmonella paratyphi A and Salmonella typhi may not produce a black centre).

 BGA agar - Colonies appear as red-pink, 1-3mm in diameter, surrounded by brilliant red zones
in the agar.

BIOCHEMICAL REACTION

Salmonellae ferment glucose, mannitol and maltose forming acid and gas. Whereas S. typhi
is an aerogenic i.e. it does not form fermentation of sugars like glucose etc. Lactose, Sucrose and
Salicin are not fermented. Indole is not produced. They are MR positive, VP negative and citrate
positive.

RESISTANCE

o o
The bacilli are killed at 55 C in one hour or at 60 C in 15 minutes. Boiling or chlorination of
water and pasteurization of milk destroy the bacilli. In polluted water it may survive for weeks and in
ice for months.

ANTIGENIC STRUCTURE

Salmonellae possess the antigens and based on which they are classified as;

(i) Flagella antigen H,

(ii) Somatic antigen O and

(iii) Surface antigen Vi

H antigen:

This antigen present on flagella is heat labile protein. It is destroyed by boiling or by treatment
with alcohols but not by formaldehyde.
 77

O antigen:

O antigen is a Phospholipid-protein-polysaccharide complex which forms an integral part of

the cell wall. It is identical with endotoxin. This is unaffected by boiling, alcohol or weak acids

CLASSIFICATION AND NOMENCLATURE

Classification within the genus is on antigenic characterisation based on Kauffman-White

scheme and this depends on identification by agglutination of the O and H antigens of the strains.
Salmonellae are classified into serological groups based on the presence of distinctive O antigen
factors and designated as 1, 2, 3 etc.

Biochemically Kauffman proposed Salmonellae classification as;

 Subgenus I: Largest and medically most important group causing human and animal
infections

 Subgenus II: Species isolated from reptiles.

 Subgenus III: Species isolated from reptiles and human beings

 Subgenus IV: These are rarely encountered.

PATHOGENESIS AND PATHOGENICITY

Although many aspects of the pathogenesis of salmonellosis are poorly understood,

particularly the relationship between salmonella toxins and cell damage, some of the general features
associated with virulence are known. The virulence of salmonellae relates to their ability to invade
host cells, replicate in them and resist both digestion by phagocytes and destruction by the
complement components of the plasma.

Following adherence, probably through fimbrial attachment, to the surface of intestinal

mucosal cells, the bacteria induce ruffling of cell membranes. The ruffles facilitate uptake of the
bacteria in membrane-bound vesicles, which often coalesce. The organisms replicate in these
vesicles and are eventually released from the cells, which sustain only mild or transient damage. The
complex invasion process is mediated by the products of a number of chromosomal genes, whereas
growth within host cells depends on the presence of virulence plasmids.

CLINICAL INFECTIONS

Salmonellosis is of common occurrence in domestic animals and the consequences of

infection range from subclinical carrier status to acute fatal septicaemia. Some Salmonella serotypes
such as Salmonella Pullorum and Salmonella gallinarum in poultry, Salmonella choleraesuis in pigs
and Salmonella Dublin in cattle are relatively host-specific. In contrast, Salmonella typhimurium has a
comparatively wide host range. It is recognised that healthy adult carnivores are innately resistant to
salmonellosis.

Salmonellae often localize in the mucosae of the ileum, caecum and colon, and in the
mesenteric lymph nodes of infected animals. Although most organisms are cleared from the tissues
by host defense mechanisms, subclinical infection may persist with shedding of small numbers of
salmonellae in the faeces. Salmonella Dublin causes a variety of clinical effects in cattle. Terminal dry
gangrene and bone lesions are common manifestations in chronic infections with Salmonella dublin
in calves.
 78

Enteric salmonellosis

Enterocolitis caused by salmonella organisms can affect most species of farm animals,
irrespective of age. Acute disease is characterized by fever, depression, anorexia and profuse foul-
smelling diarrhoea often containing blood, mucus and epithelial casts. Dehydration and weight loss
follow and pregnant animals may abort.

Septicaemic salmonellosis

In pigs with septicaemic Salmonella choleraesuis infection, there is a characteristic bluish

discolouration of the ears and snout. Intercurrent viral infections often predispose to severe clinical
forms of the disease. The close clinical and pathological relationships which have been recognized in
animals infected with Salmonella choleraesuis ('hog-cholera bacillus') and classical swine fever virus,
either jointly or separately, exemplify both the importance of intercurrent infections and the difficulty
of clinically distinguishing the diseases caused by these agents.

Salmonellosis in poultry

Salmonella pullorum, Salmonella gallinarum and Salmonella enteritidis can infect the ovaries
of hens and be transmitted through eggs. The presence of Salmonella enteritidis in undercooked egg
dishes may result in human food poisoning.

Pullorum disease or bacillary white diarrhoea (Salmonella pullorum) infects young chicks and
turkey poults up to 2 to 3 weeks of age. The mortality rate is high and affected birds huddle under a
heat source and are anorexic, depressed and have whitish faecal pasting around their vents.
Characteristic lesions include whitish nodes throughout the lungs and focal necrosis of liver and
spleen.

Fowl typhoid (Salmonella gallinarum) can produce lesions in young chicks and poults similar
to those of pullorum disease. However, in countries where fowl typhoid is endemic, a septicaemic
disease of adult birds occurs, often resulting in sudden deaths. Characteristic findings include an
enlarged, friable, bile-stained liver and enlarged spleen. As Salmonella pullorum and Salmonella
gallinarum possess similar somatic antigens. Both have been eradicated from many countries by a
serological testing and slaughter policy for pullorum disease. Paratyphoid is a name given to
infections of poultry by non-host-adapted salmonellae such as Salmonella enteritidis and Salmonella
typhimurium. These infections are often subclinical in laying birds.

DIAGNOSTIC PROCEDURES

Identification criteria for isolates:

Specimens should be cultured directly onto BG and XLD agars and also added to selenite F,
Rappaport or tetrathionate broth for enrichment and subsequent subculture. The plates and
O
enrichment broth are incubated aerobically at 37 C for up to 48 hours. Subcultures are made from the
enrichment broth at 24 and 48 hours.

On brilliant green agar, colonies and medium are red indicating alkalinity. On XLD agar,
colonies are red (alkaline) with a black centre, indicating H2S production.

Suspicious colonies, subcultured from the selective media into TSI agar and lysinc decarboxylase
O
broth, should be examined after incubation for 18 hours at 37 C to establish their biochemical identity
as salmonellae.

If reactions in TSI agar and lysine decarboxylase broth are inconclusive, a biochemical profile
using a battery of biochemical tests may allow definitive identification.
 79

The isolates from the TSI agar slant are confirmed as salmonellae using commercially
available antisera for O and H antigens in a slide agglutination test. Serotypes with O antigens in
common are assigned to a serogroup.

Serotypes which have flagellar (H) antigens in two phases, phase 1 (specific) and phase 2
(non-specific), are termed diphasic. Biotyping is required for serotypes which are antigenically
indistinguishable such as Salmonella pullorum and Salmonella gallinarum.

Phage typing is used in epidemiological studies to identify isolates with specific

characteristics such as multiple resistances to antibiotics and enhanced virulence. Examples of
important phage types are Salmonella typhimurium DT (definitive type) 104 which exhibits multiple
resistance to antibiotics and Salmonella enteritidis PT (phage type) 4 which is found in poultry
products and is a common cause of food poisoning in humans.

Serological tests such as ELISA and agglutination techniques are of greatest value when used
on a herd or flock basis. A rising antibody titre using paired serum samples is indicative of active
infection. DNA probes can be used to screen large numbers of faecal samples for salmonellae.

TREATMENT

Antibiotic therapy should be based on results of susceptibility testing because R-plasmids

coding for multiple resistance are comparatively common in salmonellae. Oral antimicrobial therapy
should be used judiciously for treating enteric salmonellosis because it may disturb the normal
intestinal flora, extend the duration of salmonella excretion and increase the probability of drug
resistance developing. In the septicaemic form of the disease, intravenous antibiotic therapy must be
used. Fluid and electrolyte replacement therapy is required to counteract dehydration and shock.

CONTROL

Control is based on reducing the risk of exposure to infection. Intensively reared, food-
producing animals are more likely to acquire infection and are also a major source of human infection.

Measures for excluding infection from a herd or flock free of salmonellosis:

 A closed-herd policy should be implemented when feasible.

 Animals should be purchased from reliable sources and remain isolated until negative for
salmonellae on three consecutive samplings.

 Stem should be taken to prevent contamination of foodstuffs and water. In this context,
rodent control is important.

 Protective clothing and footwear should be worn by personnel entering hatcheries and
minimal disease pig units.

 Measures for reducing environmental contamination:

 Effective routine cleaning and disinfection of buildings and equipment is essential.

 Overstocking and overcrowding should be avoided.

 Slurry should be spread on arable land where possible. An interval of at least two months
should elapse before grazing commences on pastures following the application of slurry.

 The continuous use of paddocks for susceptible animals should be avoided.

 80

Diseases caused by selected Salmonella serotypes

Host Salmonella serotypes Disease

Humans S. typhi Typhoid fever

S. paratyphi A Paratyphoid fever

S. schottmuelleri Paratyphoid fever

S. enteritidis, Food poisoning

S. typhimurium and
Others

Cattle S.dublin Subclinical excreters, latent carriers, enteritis,

septicaemia, meningitis in calves, abortion (with
or without other apparent clinical signs),
osteomyelitis, joint ill, terminal dry gangrene in
calves

S. typhimurium, Enteritis or septicaemia

S. bovismorbificans
and others

Pigs S. choleraesuis and Severe outbreaks clinically similar to swine lever

S. choleraesuis (hog cholera), and swine fever can be followed by
biotype Kunzendorf a secondary infection with S.choleraesuis,
earning it the name of 'hog cholera bacillus'.
Rectal stricture is sometimes a sequel of the
disease

S. typhisuis Chronic enteritis in young pigs. Far less virulent

than S. Choleraesuis

S. typhimurium and Enteritis or septicaemia

Others

Sheep S. abortusovis Abortion in ewes. S. abortusovis is present in

S. montevideo Britain, Europe and the Middle East
S.dublin

S. typhimurium, Enteritis or septicaemia

S. anatum and others

Horses S. abortusequi Abortion in mares. The serovar is present in

Europe, South Africa and South America but is
now rare in the USA

S. typhimurium and Enteritis or septicaemia, especially in foa ls and

others stressed adults

Poultry and other S.pullorum Pullorum disease (bacillary white diarrhoea) in

birds chicks. Transovarian transmission

Fowl typhoid in all ages, mainly adults.

S. gallinarum Egg transmitted
 81

Severe infections (enteritis and septicaemia) in

S. arizonae chicks and turkey poults. Egg transmitted.
Serovar associated with reptiles. Occasional
infections in other animals

Collectively known as 'fowl paratyphoid'.

Inapparent infections, enteritis and septicaemia.
S. enteritidis, S. enteritidis may be egg transmitted.
S. typhimurium and
many other serotypes S. typhimurium can cause sudden deaths
(septicaemia) in pigeon squabs, or if they survive,
swollen wing joints

YERSINIA

Yersinia species are non-lactose fermenters and, with the exception of Y.pestis are motile.
Although there are more than 10 Yersinia species, only Y.pestis, Y.enterocolitica and
Y.pseudotuberculosis are pathogenic for animals and man. Yersinia ruckeri causes perioral
haemorrhagic inflammation in some species of fish. Growth of yersiniae tends to be less rapid than
other members of the Enferobacteriaceae. They characteristically demonstrate bipolar staining in
Giemsa-stained smears from animal tissues
 82

NATURAL HABITAT

Y. pestis, the cause of bubonic plague in man and a sylvatic cycle in animals, is transmitted
mainly by fleas from tolerant rodents. Human infections through cuts, bites, scratches and aerosols
can also occur. Cats are susceptible to Y.pestis and naturally infected cats can pose a health hazard
for humans in endemic areas. Y.pseudotuberculosis persists in wild rodents and birds as well as in
the environment. The intestinal tract of wild and domestic animals appears to be the reservoir for Y.
enterocolitica. Pigs, particularly, are carriers of Y. enterocolitica strains pathogenic for humans.

ISOLATION

Yersinia species grow on nutrient, blood and MacConkey agars but the colonies, after 24
hours incubation, tend to be smaller than those of the other members of the Enterobacteriaceae. Y.
pestis grows poorly on agars containing desoxycholate whereas Y.enterocolitica and
Y.pseudotuberculosis grow well on these media. Yersinia selective medium (CIN agar) containing the
antibiotic supplement cefsulodin (15mg/litre), irgasin (4 mg/litre) and novobiocin (2.5 mg/litre) is
designed for the isolation of Y. enrerocolitica from faeces.

A cold-enrichment procedure may be necessary for the isolation of both Y. enterocolitica and
Y.pseudotuberculosis from faecal specimens. A faecal specimen (approximately 5 per cent by volume)
is placed in1/15M phosphate buffered saline (Oxoid) and held in the refrigerator (4 °C) for 3 weeks.
Subcultures, at weekly intervals, can be made on MacConkey and Yersinia selective medium. Yersinia
species usually grow faster at 37°C but prefer lower incubation temperatures, particularly on primary
isolation. Sometimes additional culture plates, incubated at 22-25°C, can be useful for initial isolation.

COLONIAL MORPHOLOGY

The Yersinia species are lactose-negative although lactose-positive strains of Y.

enterocolitica occur and this property is thought to be plasmid-mediated. Y. enterocolitica will grow
well on media, such as brilliant green and XLD agar intended for salmonella isolation but
Y.pseudotuberculosis is less tolerant. Y.enerocolitica and Y. pseudotuberculosis are non-haemolytic
on blood agar. The colonies of Y. enterocolitica on Yersinia selective medium (Oxoid) have dark red
centres with a transparent periphery.

BIOCHEMICAL TESTS

Y. pestis, Y.pseudotuberculosis and Y. enterocolitica are nonmotile at 37°C but Y.

pseudotuberculosis and Y.enterocolitica strains can be motile at 28°C. Y. pseudotuberculosis is
almost always urease-positive as are most strains of Y. enterocolitica, but Y. pestis does not produce
this enzyme. They are negative for the pyrazinamidase test, salicin fermentation and aesculin
hydrolysis, whereas the non-pathogenic strains are positive to these tests. The pathogenic strains
grow as small red colonies on congored-magnesium oxalate (CR-MOX) agar but non-pathogenic
strains are unable to grow on this medium.

ANTIGENS OF YERSINIA SPECIES

Y. pseudotuberculosis can be divided into 6 serogroups based on the thermostable O

antigens (I-VI) and five H (flagella) antigens (a-e). There are shared antigens between the closely
related Y.pestis and Y.pseudotuberculosis. An antigenic relationship exists between
Y.pseudotuberculosis and salmonella O antigens in serogroups B, D and E and also with some E.coli
O antigens. Y.enterocolitica shares an antigen with Brucella species that can give rise to false brucella
agglutination test results. Y.enterocolitica is divided into more than 37 serotypes, many of which do
not appear to be pathogenic.
 83

PATHOGENESIS AND PATHOGENICITY

Pathogenic yersiniae are facultative intracellular organisms which possess plasmid and
chromosomal encoded virulence factors, many of which are required for survival and multiplication in
macrophages. Yersinia pseudotuberculosis and Y.enterocolitica are less virulent than Y.pestis and
rarely produce generalized infections. The pathogenetic mechanisms in enteric disease caused by
Y.enterocolitica and Y. pseudotuberculosis are incompletely understood. It is probable that both
organisms gain entry to the mucosa through M cells of Peyer's patches. Adhesion to and subsequent
invasion through these cells are facilitated by factors such as invasion and adhesion / invasion
proteins which have an affinity for integrins on cell surfaces. Once in the mucosa, the bacteria are
engulfed by macrophages in which they survive and are transported to the mesenteric lymph nodes.
Replication in the nodes follows with the development of necrotic lesions and neutrophil infiltration.
Survival of Y.pseudotuberculosis and Y.enterocolitica is enhanced by antiphagocytic proteins
secreted by the organisms which interfere with the normal functioning of neutrophils in the host.

Yersinia pestis is more invasive than Y.pseudotuberculosis and Y. enterocolitica and

possesses additional virulence factors. These include an antiphagocytic protein capsule (Fraction 1)
and a plasminogen activator which aids systemic spread. Endotoxin, with properties similar to the
endotoxin produced by other members of the Enterobacteriaceae, also contributes to the
pathogenesis of disease.

CLINICAL INFECTIONS

Yersinia pseudotuberculosis causes enteric infections, in a wide variety of wild and domestic
animals which are often subclinical. The septicaemic form of disease, known as pseudotuberculosis,
can occur in laboratory rodents and aviary birds. Sporadic abortions caused by Y.pseudotuberculosis
have been reported in cattle, sheep and goats.

Wild and domestic animals may act as reservoirs of Yersinia enterocolitica which is primarily
a human enteric pathogen. The pig is the natural reservoir for Y. enterocolitica serotype 03 biotype 4,
which is an important pathogen in humans. Rare cases of enteric disease, precipitated by stress, may
be encountered in pigs, farmed deer, goats and lambs. Yersinia enterocolitica has been implicated in
sporadic ovine abortion. Yersinia pestis, the cause of human bubonic plague ('black death'), can infect
both dogs and cats in endemic areas. Cats, which are particularly susceptible, may be a source of
infection for owners and attending veterinarians.

Enteric yersiniosis

Enteritis caused by Y.pseudoruberculosis is relatively common in young farmed deer. Enteric

disease has been reported in sheep, goats and cattle less than one year of age. Subclinical infection
in many species is common and clinical disease may be precipitated in the winter months by stress
factors such as poor nutrition, weaning, transportation and cold wet conditions. There may be
prolonged survival of Y. pseudotuberculosis on pasture in cold wet weather, facilitating faecal-oral
transmission.

Enteritis in young deer and lambs is characterized by profuse watery diarrhoea, sometimes
blood-stained, which may be rapidly fatal if untreated. The luminal contents of the small and large
intestine are watery and mucosal hyperaemia is evident at postmortem examination. Severely
affected animals may show mucosal ulceration. The mesenteric lymph nodes are often enlarged and
oedematous and scattered pale necrotic foci may be present in the liver. A clinically similar but less
severe enterocolitis caused by Yersinia enterocolitica has described in young ruminants.

Diagnosis

The species and age group affected, especially during cold wet spells of weather, may
 84

suggest yersiniosis. Histological examination of intestinal lesions may reveal clusters of organisms in
micro-abscesses within the mucosa. Confirmation requires isolation and identification of
Y.pseudotuberculosis or, occasionally, Y. enterocolitica:

 Samples from tissues can be plated directly onto blood and MacConkey agars and incubated
aerobically at 37°C for up to 72 hours.

 Faecal samples may be plated directly onto special selective media.

 A cold enrichment procedure may facilitate recovery of yersiniae from faeces especially if
they are present in low mumbers. A 5% suspension of faeces in phosphate buffered saline,
held at 4°C for three weeks, is subcultured weekly onto MacConkey agar.

 Serotyping may be necessary to establish whether the isolates belong to known pathogenic
serotypes.

Treatment and control

Fluid replacement therapy together with broad spectrum antimicrobial treatment should be
initiated promptly in young animals.

A formalin-killed Y.pseudotuberculosis vaccine composed of serotypes I, II and III,

administered in two doses three weeks apart has been shown to decrease the occurrence of clinical
disease in young deer,

Stressful conditions should, where practicable, be minimized.

DISEASES CAUSED BY YERSINIA SPECIES

Yersinia species Host Disease(s)

Y. pestis Humans Bubonic plague ('black death')

Rodents Sylvatic plague. Infection in

rodents usually latent with
occasional outbreaks of
disease

Cats Cats in endemic areas may show mandibular

lymphadenitis, fever, depression, anorexia, sneezing
and occasionally nervous disturbances.
Most infections are fatal

Y.pseudotuberculosis Guinea-pigs, Pseudotubercu losis

other rodents, 1. Septicaemic syndrome. 2 Classical syndrome with
rabbits, wild nodules in internal organs Seen most commonly in
and captive guinea-pigs and canaries
birds

Farm animals
Latent infections, Occasional disease such as in captive
deer
Sheep
Orchitis and epididymitis reported
Humans
Mesenteric lymphadenitis, acute terminal ileitis and rare
cases of septicaemia. Mainly children and young adults
 85

Y. enterocolitica Farm animals Latent infections with sporadic cases of enteritis or

generalised infections. Captive deer particularly
susceptible

Humans Food poisoning (enteritis), mesenteric lymphadenitis

(pseudo-appendicitis)
Most common in children

PROTEUS AND PROVIDENCIA

The genera Proteus and Providencia belong to the tribe Proteae of the family
Enterobacteriaeceae. The members of both these genera are gram negative, motile bacilli, aerobes
and facultative anaerobes and can grow on basic media. A characteristic feature which distinguishes
tribe Proteae from other members of Enterobactereaceae is the presence of the enzyme
phenylalanine-deaminase which converts phenylalanine to phenylpyruvic acid (PPA reaction).They
also produces a powerful urease enzyme which rapidly hydrolyses urea to ammonia.

GENUS PROTEUS

They are gram-negative bacilli, 1-3 μm long and 0.6 μm wide. They are noncapsulated and are
actively motile by peritrichous flagella. The name ‘Proteus’ refers to their pleomorphism, after the
Greek God Proteus who could assume any shape. Four species: Proteus mirabilis, P.vulgaris,
P.penneri and P.myxofaciens are recognized. Proteus mirabilis, P.vulgaris are widely recognised as
human pathogens.

CULTURE CHARACTERISTICS

These can grow on ordinary media like nutrient agar with a characteristic fishy or seminal
odour. On MacConkey and Teepol lactose agar, lactose non-fermenting pale colonies, around 2-3 mm
in size are formed. On non-inhibitory solid media such as blood and nutrient agar Proteus mirabilis
and P vulgaris show characteristic swarming growth in the form of a uniform film, which spreads over
the whole surface of the plate. In young swarming cultures, many of the bacteria are long, curved and
filamentous, sometimes reaching upto 80 μm in length. When two different strains of swarming
proteus mirabilis encounter one another on an agar plate, swarming ceases and a visibile line of
demarcation forms. This is known as the Dienes phenomenon.

In liquid medium (peptone water, nutrient broth), Proteus produces uniform turbidity with a
 86

slight powdery deposit and an ammoniacal odour.

Swarming inhibitory methods: Swarming of Proteus can be prevented by;

 Increasing the concentration of agar from 1-2% to 6%.

 Incorporation of sodium azide, boric acid, or chloral hydrate.

 Introducing growth inhibitors like sulphonamides.

 On Teepol Lactose agar by Teepol (surface active agent)

 On MacConkey agar or DCA by presence of bile salts.

 On CLED agar by the absence of electrolytes.

BIOCHEMICAL REACTIONS

Like all other members of the family Enterobacteriaceae, all the species of genus Proteus are
catalase positive, oxidase negative, reduce nitrates to nitrites and show fermentative reaction on
Hugh Leifson’s of media. All members of the tribe Proteeae are PPA positive and, hydrolyse urea to
ammonia which differentiates them from other Enterobacteriaeceae.

ANTIGENIC STRUCTURE

The bacilli possess thermostable ‘O’ antigen (somatic) and thermolabile ‘H’ (flagellar) antigen.
Weil and Felix observed that certain non-motile strains of P.vulgaris, called the ‘X’ strains, were
agglutinated by sera from patients with typhus fever. This heterophile agglutination due to the sharing
of carbohydrate antigen by certain strains of Proteus and Rickettsia forms the basis of the Weil-Felix
reaction and is used for diagnosing rickettsial infections. Three non-motile Proteus strains OX-2 and
OX-19 of P vulgaris and OX-K of P.mirabilis are used in the agglutination test.

TYPING METHODS

 Serotyping

 Phage typing

 Dienes typing

Bacteriocin (proticin) typing

Dienes phenomenon: This method forms the basis of typing swarming strains of Proteus for
local epidemiological studies. Different cultures are inoculated as discrete spots on the same plate
and allowed to swarm towards one another. A line of complete or partially inhibited growth is formed
where cultures of different strains meet; no line is formed between cultures of the same strain.

PATHOGENECITY

Proteus species are saprophytic and widely distributed in nature. They also occur as
commensals in the intestine. They are opportunistic pathogens and may cause many types of
infections such as:

Urinary tract infections (UTI) with predilection for upper UTI:

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It produces urease which liberates ammonia from urea. The alkaline conditions lead to the
precipitation of phosphates and the formation of calculi in the urinary tract.

Proteus species may also be associated with the following conditions;

 Pyogenic lesions

 Wound infections

 Otitis media

 Meningitis

 Septicaemia

 Osteomyelitis

LABORATORY DIAGNOSIS

(i) Specimen:

 UTI- midstream urine

 Wound/ abscesses, osteomyelitis, otitis media: pus

 Meningitis: CSF

 Septicaemia – blood culture

(ii) Culture: Clinical specimens should be cultured on MacConkey agar/Teepol lactose agar, 6% blood
agar and in case of urine on CLED (Cysteine lactose electrolyte deficient agar). Culture media are
incubated at 37ºC for 18-24 hours. Pale coloured Non-Lactose Fermenting (NLF) colonies are seen on
MacConkey agar. Identification is done by standard biochemical reactions.

(iii) Antibiotic susceptibility: Proteus are resistant to many of the common antibiotics, except P
mirabilis which is sensitive to ampicillin and cephalosporins but nitrofurantoin is not effective.

GENUS PROVIDENCIA

Like Proteus, strains of Providencia are Non-lactase fermenting (NLF), methyl red and PPA
positive bacilli which are motile by peritrichous flagella. However, they do not swarm on solid media.
They can often be recognized by their ‘fruity’ smell. Three important pathogenic species include
Prov.alcalifaciens, Prov.rettgeri and Prov.stuartii. It has been suggested that Prov alcalifaciens causes
diarrhea, Prov.rettgeri and Prov. stuartii have been associated with urinary-tract, wound and other
infections. Providencia are very resistant to antibiotics, particularly Prov.stuartii which is also
resistant to disinfectants, making it a major pathogen in burn units.
 88

PSEUDOMONAS

HISTORY

Pseudomonas is comprised of two Greek words-Pseudo meaning false and Monas meaning
unit, so literally the term means “false unit”. However Pseudomonas is a real bacteria so we really do
not know the basis of the nomenclature by Walter Migula. A scientist by the name of Migula gave the
Genus name of Pseudomonas in 1894 to these bacteria.

CLASSIFICATION

The classification of Pseudomonas is given below:

 Class : Gamma Proteobacteria

 Order : Pseudomonadales

 Family : Pseudomonadaceae

 Genus : Pseudomonas

 Eight groups : P. aeruginosa ; P. chlororaphis; P. fluorescens; P.pertucinogena;

P.putida; P. stutzeri; P. syringae; P. incertae sedis.

The family has 191 valid species. These include bacteria which are saprophytic, free living,
and human, animal and plant pathogens. The type species is Pseudomonas aeruginosa.

USUAL HABITAT

Pseudomonas species are environmental organisms which occur worldwide in water and soil,
 89

and on plants. Pseudomonas aeruginasa is also found on the skin, on mucous membranes and in
faeces. Burkholderia pseudomallei, which is found in soils, occasionally infects animals and man.
Wild rodents can act as reservoirs of this organism. It is widely distributed in some tropical and
subtropical regions of Southeast Asia and Australia. Although B.mallei can survive in the environment
for up to 6 weeks its reservoir is infected Equidae.

CLINICAL INFECTIONS

Burkhlderia mallei a major pathogen of Equidue causes both acute and chronic disease. It
manifests mainly as lesions in the skin and the respiratory tract, Infection with B.pseudomallei can
cause chronic suppurative lcsions in the lungs and other organs of a wide range of species. In
contrast, P.aeruginosa is an opportunistic pathogen which may occasionally cause acute systemic
disease.

MORPHOLOGY

Pseudomonas is rod shaped, slender (0.5 to 0.8 μm by 1.5 to 3.0 μm) Gram negative
organism, occurs as single bacteria, in pairs, and occasionally in short chains. motile by polar flagella,
sometimes more than two flagella may be present. Some strains of Pseudomonas particularly those
isolated from cases of cystic fibrosis are very mucoid and have kind of pseudo capsule (glycocalyx)
made of polysaccharides. The glycocalyx protects pseudomonas from host defense.

Pseudonlonas aeruginosa, Burkholderia mallei and B.pseudomallei are Gram-negative rods

(0.5 to 1.0 x 1 to 5 µm) which are obligate aerobes and oxidize carbohydrates. Most isolates are
oxidase-positive and catalase positive. They are motile by one or more polar flagella, with the
exception of B.mallei which is non-motile. The majority of these organisms have no special growth
requirements and grow well on MacConkey agar. Burkholderia mallei require 1% glycerol in media for
optimal growth. Pseudomonas aeruginosa, characterized by the production of diffusible pigments,
causes a variety of opportunistic infections in a wide range of animals.

A number of other Pseudomonas species may be isolated from clinical specimens.

Pseudomonass fluorescens and P.putida occasionally infect freshwater fish. Burkholderia species,
previously classified in the genus Pseudomnonas, include B. mallei, the cause of glanders and
B.psoudomallei, the cause of melioidosis. Both diseases are zoonoses.

Culture

The Pseudomonas species are non-fastidious and will grow on trypticase soy agar, blood
agar and on less complex media. The growth of P. mallei is enhanced by 1 per cent glycerol. A
selective medium for P.mallei can be made by adding 1000 units polymyxin E, 1250 units bacitracin
and 0.25 mg actidione to 100 ml of trypticase soy agar. Commercial selective media are available for
P. aeruginosa and usually contain 0.03 per cent cetrimide (cetyl trimethyl ammonium bromide).

P.aeruginosa will also grow on many of the selective media intended for the
Enterobacteriaceae such as MacConkey, brilliant green and XLD agars.

The cultures for P.aeruginosa, P.pseudomallei and P.mallei are incubated aerobically at 37°C
for 24 - 48 hours. Some of the saprophytic pseudomonads, such as P.fluorescens, grow extremely
poorly, or not at all, at 37°C as 30°C is often the upper temperature limit of their growth range.

P. aeruginosa is an obligate aerobe that grows readily on many types of culture media,
sometimes producing a sweet or grapelike or corn taco–like odor. Some strains hemolyze blood. P
aeruginosa forms smooth round colonies with a fluorescent greenish color. It oft en produces the
nonfl uorescent bluish pigment pyocyanin, which diff uses into the agar. Other Pseudomonas species
do not produce pyocyanin.
 90

Many strains of P.aeruginosa also produce the fluorescent pigment pyoverdin, which gives a
greenish color to the agar. Some strains produce the dark red pigment pyorubin or the black pigment
pyomelanin.

P.aeruginosa in a culture can produce multiple colony types. P aeruginosa from different
colony types may also have different biochemical and enzymatic activities and different antimicrobial
susceptibility patterns. Sometimes it is not clear if the colony types represent different strains of P.
aeruginosa or are variants of the same strain.

P.aeruginosa grows well at 37–42°C; its growth at 42°C helps differentiate it from other
Pseudomonas species in the fluorescent group. It is oxidase positive. It does not ferment
carbohydrates, but many strains oxidize glucose. Identification is usually based on colonial
morphology, oxidase positivity, the presence of characteristic pigments, and growth at 42°C.

Pseudomonas is a strict (obligate) aerobe, but sometimes it can grow anaerobically if nitrates
(NO3 act as respiratory electron acceptor) are present in the medium.

Pseudomonas can grow at wide ranges of temperature; the optimum temperature is 37°C. It
can grow on ordinary media like nutrient agar and grows almost on all the culture media used
routinely in the bacteriology lab. Pseudomonas has been seen to grow in distilled water also.

Pseudomonas produces large, opaque, flat colonies with irregular margins and distinctively
fruity odour colonies. The colour of growth will depend upon the type of pigments (enumerated below)
produced by the organism. The isolates from water and soil produce small round colonies. The
isolates from clinical specimens like respiratory, urine, etc. may produce mucoid colonies. The
bacteria which form mucoid colonies are more virulent compared to others.

The pigments produced by Pseudomonas are:

 The fluorescent pigment pyoverdin (greenish yellow)

 The blue pigment pyocyanin (bluish green)

 Pyorubin (red)

 Pyomelanin (brown)

COLONIAL MORPHOLOGY

P.aeruginosa: The colonies are large (3-4 mm), flat, greyish-blue with a characteristic fruity,
grapelike odour of aminoacetophenone. Most strains give a clear zone of haemolysis on blood agar.
Pyocyanin, a bluish pigment unique to P.aeruginosa, gives the blue colour associated with many
cultures. Colonial variation includes S-forms (soft and shiny), R-forms (dry and granular) that are not
unlike (he colonies of some Bacillus species, and mucoid M-forms that are frequently biochemically
atypical. Some strains have colonies with a distinctive metallic sheen. P. aeruginosa produces large,
pale colonies on MacConkey agar (unable to utilise lactose) with greenish-blue pigment
superimposed. Red colonies and medium, indicative of an alkaline reaction, are seen on brilliant green
and XLD agars. No H2S is produced on XLD medium.

P. pseudomallei: Colonial growth varies from smooth and mucoid to rough with a dull,
wrinkled, corrugated surface. In the smooth form the colonies are round, low-convex, entire, shiny and
greyish-yellow. After several days the colonies become opaque, yellowish-brown and umbonate. The
growth has a characteristic earthy or musty odour. Partial and, later, complete haemolysis occurs on
sheep blood agar. P. pseudomallei grow on MacConkey agar, utilising lactose, but there is no growth
on deoxycholate or Salmonella-Shigella (SS) agars.
 91

P. mallei: growth is slower than that of P.aeruginosa and P.pseudomallei but in 24-48 hours
the colonies are 1- 2 mm in diameter, smooth and white to cream. As they age, they become granular
and yellowish or brown in colour. P.mallei is unable to grow on MacConkey agar.

The pigments of P. aeruginosa;

Strains of P. aeruginosa produce the diffusible pigments pyocyanin (blue). pyoverdin (yellow),
pyorubin (red) and pyomelanin (dark brown) in varying combinations and amounts. Some strains
produce all four pigments. Pyorubin and pyomelanin are less commonly produced, develop slowly and
are seen best by growing the strains on nutrient agar slants at room temperature for up to 2 weeks.
As pyocyanin is unique to P. aeruginosa this is an important diagnostic characteristic although strains
vary in the amount of the pigment they produce. Media such as Pseudomonas agar P (Difco) will
enhance pyocyanin production and Pseudomonas agar F (Difco) enhances pyoverdin production.
Pyoverdin, once called ' fluorescein', will fluoresce under ultra- violet light. Some strains of P.
aeruginosa do not produce pyocyanin and these may also be atypical in certain biochemical reactions,
making them difficult to identify.

BIOCHEMICAL CHARACTERISTICS

 Pseudomonas has oxidative metabolism. Since organism is non fermentative the acid is not
produced from peptone water sugars.

 The important biochemical characteristics of Pseudomonas include:

 Oxidase test positive;

 Catalase test positive

 Nitrates are reduced to nitrites;

 Arginine dihydrolase test positive;

 Glucose is utilized oxidatively ⇒ Oxidative reaction in of media

 Indole, Methyl red (MR), Vogues Prauskar (VP) and H2S production test are negative.

 Commonest screening diagnostic biochemical test used in lab is the oxidase test.

ANTIGENIC STRUCTURE AND TOXINS

Pili (fimbriae) extend from the cell surface and promote attachment to host epithelial cells.
The exopolysaccharide is responsible for the mucoid colonies. The lipopolysaccharide, which exists in
multiple immunotypes, is responsible for many of the endotoxic properties of the organism.
P.aeruginosa can be typed by lipopolysaccharide immunotype and by pyocin (bacteriocin)
susceptibility.

Most P.aeruginosa isolates from clinical infections produce extracellular enzymes, including
elastases, proteases, and two hemolysins (a heat-labile phospholipase C and a heat-stable glycolipid).

Many strains of P.aeruginosa produce exotoxin A, which causes tissue necrosis and is lethal
for animals when injected in purified form. The toxin blocks protein synthesis by a mechanism of
action identical to that of diphtheria toxin, although the structures of the two toxins are not identical.

VIRULENCE AND PATHOGENICITY

Pseudomonas can infect any tissue, any organ system in an immune-compromised host. P.
 92

aeruginosa produces exotoxin A which is a virulence factor. This exotoxin inactivates ADP ribosylate
eukaryotic elongation factor 2 (EF2) and thus interferes with the synthesis of protein resulting in
death of the cell. P.aeruginosa also produces an exoenzyme “Exo A” which damages the cell
membrane leading to lysis of the membrane and cell death.

The most important risk factor for Pseudomonas infections is break down of host defense
due to disease or other factors. Pseudomonas is both invasive and toxinogenic. Infection involves the
following three steps:

 Bacterial attachment and colonization;

 Local invasion;

 Disseminated systemic disease.

PATHOGENESIS

P.aeruginosa is pathogenic only when introduced into areas devoid of normal defenses, such
as when mucous membranes and skin are disrupted by direct tissue damage as in the case of burn
wounds.

The bacterium attaches to and colonizes the mucous membranes or skin, invades locally, and
produces systemic disease. These processes are promoted by the pili, enzymes, and toxins described
earlier. Lipopolysaccharide plays a direct role in causing fever, shock, oliguria, leukocytosis and
leukopenia, disseminated intravascular coagulation, and respiratory distress syndrome.

P.aeruginosa and other pseudomonads are resistant to many antimicrobial agents and
therefore become dominant and important when more susceptible bacteria of the normal microbiota
are suppressed.

Bacterial attachment and Colonization

Pseudomonas infection may be endogenous (may be from intestines) or may be acquired

from outside (exogenous). The adhesins are the pili of P aeruginosa with which bacteria adhere to
specific galactose or mannose or sialic acid receptors on the mucosal epithelial cells of the upper
respiratory tract and others. Production of protease enzyme by bacteria breaks down the fibronectin
and exposes the pilus specific receptors on the epithelial cell surface.

Tissue injury caused by viral infection and other phenomenon facilitates colonization by
Pseudomonas (Opportunistic colonization). Pseudomonas can also colonize by formation of biofilm
which we will discuss later. Pseudomonas pili, mucoid polysaccharide, probably surface-bound
exoenzyme S and possibly other cell surface adhesins help Pseudomonas to colonize.

Pseudomonas can invade the blood stream from initial site of infection and through blood is
disseminated to different organs. The factors which help bacteria to invade as described above help
to invade the organs, tissues wherever the bacteria reach. Bacterial endotoxin during septicaemia,
may cause fever, hypotension, and intravascular coagulation.

The virulence/pathogenicity armamentarium of Pseudomonas includes:

 Adhesins

 Pili (N-methyl-phenylalanine pili)

 Polysaccharide capsule (glycocalyx)

 93

 Slime

 Invasins

 Elastase

 Alkaline protease

 Hemolysins (phospholipase and lecithinase)

 Cytotoxin (leukocidin)

 Pyocyanin

 Toxins

 Exoenzyme S

 Exotoxin A

 Lipopolysaccharide (LPS)

 Antiphagocytic elements

 False capsules, slime layer

 Lipo polysaccharide

 Biofilm formation

PATHOGENESIS AND PATHOGENICITY

Pathogenic strains of P.aeruginosa produce a variety of toxins and enzymes which promote
tissue invasion and damage. Attachment to host cells is mediated by fimbriae. Colonization and
replication are aided by antiphagocytic properties of exoenzyme S, extracellular slime and outer
membrane lipopolysaccharides. Resistance to complement-mediated damage and the abiIity to
obtain iron from host tissues are additional virulence factors.

Tissue damage is caused by toxins such as exvtoxin A, phospholipase C and proteases.

Exotoxin A is a bipartite toxin with binding and active components. The active component, once
internalized in a cell blocks protein synthesis by ADP-ribosy lation and elongation of Factor 2 with
resultant cell death. The cytoplasmic membranes of neutrophils are damaged by a leukocidin.
Dissemination is aided by exoenzyme S and systemic toxicity is attributed to exotoxin A and
endotoxin. The host defence mechanisms against P.aeruginosa include opsonizing antibodies and
phagocytosis by macrophages.

DIAGNOSTIC PROCEDURES

 Specimens for laboratory examination include pus, respiratory aspirates, mid-stream urine,
mastitic milk and ear swabs.

 Blood agar and MacConkey agar plates, inoculated with suspected material are incubated
aerobically at 37°C for 24 to 48 hours.

Identification criteria for isolates:

 Colonial morphology are the characteristic fruity, grape-like odour

 94

 Pyocyanin production

 Lactose-negative, pale colonies on MacConkey agar

 Oxidase-positive

 Triple sugar iron agar unchanged

 Biochemical tests

TREATMENT AND CONTROL

Predisposing causes and sources of infection should be identified and, where possible,
eliminated. Pseudomunus aeruginosa is extremely resistant to many antibiotics and susceptibility
testing should be carried out on isolates. A combination of either gentamicin or tobrarnycin with
either carbenicillin or ticaricillin may be effective. Vaccines may be required for farmed mink and
chinchillas. As there arc antigenic differences between strains, polyvalent or autogenous formalin-
killed bacterins should he employed. Humoral antibody induced by a polyvalent exotoxin A-
polysaccharide vaccine appears to be protective.

GLANDERS

Glanders, caused by B. mallei, is a contagious disease of Equidae characterized by the

formation of nodules and ulcers in the respiratory tract or on the skin. Humans and carnivores are
also susceptible to infection.

Pathogenesis

Glanders in the horse is usually a chronic, disseminated, debilitating disease but the
mechanisms of pathogenicity are not known. The presence of B.mallei in the host gives rise to a
hypersensitivity reaction, the basis of the mallein test.

Transmission follows ingestion of food or water contaminated by nasal discharges of

infected Equidae. Less commonly, infection may be acquired by inhalation or through skin abrasions.
An acute septicaemic form of the disease is characterized by fever, mucopurulent nasal discharge
and respiratory signs. Death usually follows within a few weeks. Chronic disease is more common
and presents as nasal, pulmonary and cutaneous forms, all of which may be observed in an affected
animal.

In the nasal form, ulcerative nodules develop on the mucosa of the nasal septum and lowcr
part of the turbinates. A purulent, blood-stained nasal discharge and regional lymphadenopathy are
usually present. The ulcers eventually heal leaving star-shaped scars. The respiratory form i s
characterized by respiratory distress and the development of tubercle-like lesions throughout the
lungs. The cutaneous form, termed farcy, is a lymphangitis in which nodules occur along the course
of the lymphatic vessels of the limbs. Ulcers develop and discharge yellowish pus.

Chronically affected animals may die after several months or may recover and continue to
shed organisms from the respiratory tract or skin. Carnivores may contract the disease by eating
infected carcases.

Diagnostic procedures
 95

In regions where the disease is endemic, clinical signs may be diagnostic. Specimens for
laboratory diagnosis should include discharges from lesions and blood for serology. Specimens must
be processed in a biohazard cabinet.

Burkholderia mallei grow on media containing 1% glycerol and most strains will grow on
MacConkey agar. Plates are incubated aerobically at 37'C for 2 to 3 days.

Identification criteria for isolates:

 Colonial characteristics

 Majority of strains grow on MacConkey agar without utilizing lactose

 Comparatively unreactive biochemically and non-motile

Immunological tests

Suitable serological tests include the complement fixation test and agglutination techniques.

Mallein test

The mallein test is an efficient field test both for confirmation and for screening in-contact
animals. Mallein, a glycoprotein extract of P.mallei, is injected intradermally (0.1 ml) just below the
lower eyelid. A positive reaction is indicated by local swelling and mucopurulent ocular discharge after
24 hours.

Mallein tests are used to demonstrate the hypersensitivity developed after infection with P. mallei.
Mallein is a glycoprotein extracted from the bacterium. In infected animals, subcutaneous inoculation
of mallein (subcutaneous test) results in swelling at the injection site and fever. Instillation of mallein
into the conjunctival sac (ophthalmic test) is followed in 6-12 hours by an inflammatory and purulent
reaction in the eye, whereas inoculation of a small amount of mallein into the skin of the lower eyelid
(intrapalpebral test) gives a localised, oedematous swelling and purulent conjunctivitis. Healed
lesions of the nasal mucosa are activated in glanderous animals after mallein tests and this can be a
useful diagnostic feature.

Animal inoculation

The Straus reaction is seen in male guinea-pigs inoculated intraperitoneally with infective
material containing either P.pseudomallei or P.mallei. A localised peritonitis and a purulent
inflammation of the testicular tunica vaginalis develops in 2-3 days.

Antibiotic susceptibility tests

This need to be carried out with P.aeruginosa isolates as multiple drug resistance, associated
with R factors, is frequently encountered with this bacterium.

Treatment and control

A test and slaughter policy is enforced in countries where the disease is exotic. In endemic
areas, antibiotic therapy is inappropriate as treated animals often become subclinical carriers.
Effective cleaning and disinfection of all contaminated carcass must be carried out. Formalin (1.5%)
or an iodophor (2.0%) can be used, with a contact time of 6 hours.

MELIOIDOSIS
 96

Melioidosis, caused by B.pseudomellei, is endemic in tropical and subtropical regions of

southeastern Asia and Australia where rhe organism is widely distributed in soil and water. Infection
may follow ingestion, inhalation or skin contamination from environmental sources. The bacterium is
an opportunistic pathogen and stress factors or immunosuppression may predispose to clinical
disease.

Many animal species, including humans, are susceptible and subclinical infections may occur.
Because infection is usually disseminated, abscesses develop in many organs including lungs, spleen,
liver, joints and central nervous system. Melioidosis is a chronic, debilitating, progressive disease,
often with a long incubation period. Clinical signs, which are variable, relate to lesion severity and
distribution. In horses melioidosis, which can mimic glanders, is often referred to as pseudoglanders.

Pathogenesis and pathogenicity

The pathogenesis of melioidosis is poorly understood. Extracellular products of B.

psendomallei such as an exotoxin, a dermonecrotic protease and a lecithinase have been implicated
in disease production. Both strain virulence and host immunosuppression may influence the
establishment and outcome of infection.

Diagnostic procedures

 In regions where the disease is encountered, gross pathological findings may aid diagnosis.

 Specimens for laboratory diagnosis should include pus from abscesses, affected tissues and
blood for serology.

 A biohazard cabinet must be used for processing specimens.

 A fluorescent antibody technique for demonstrating the organism in tissue smears is

available in some reference laboratories.

 Blood agar and MacConkey agar plates, inoculated with suspected material, are incubated
aerobically at 37°C for 24-48 hours.

 Identification criteria for isolates:

- Colonial morphology and characteristic musty odour

- Lactose utilized in MacConkey agar

- Biochemical characteristics

 Slide agglutination test using specific antiserum

 ELISA, complement fixation and indirect haemagglutination tests can be used for detecting
serum antibodies.

Treatment and control

 Confirmation of infection followed by slaughter of infected animals is mandatory in countries

where the disease is exotic.

 Treatment is expensive and unreliable. Relapses can occur after antibiotic therapy is
discontinued.
 97

 Vaccines are being developed in some countries.

DISEASES AND MAIN HOSTS OF THE PATHOGENIC PSEUDOMONAS SPECIES

Species Host(s) Disease

P. aeruginosa Cattle Mastitis, uterine infections, skin infections,

abscesses, enteritis and arthritis

Sheep and goats Mastitis, pneumonia, lung abscesses and 'green wool'
(a skin infection in sheep)

Pigs Enteritis, respiratory infections and otitis

Horses Metritis, lung abscesses and eye infections

Dogs and cats Otitis externa, cystitis, endocarditis, dermatitis, wound

infections and conjunctivitis

Mink Septicaemia and pneumonia

Chinchilla Generalised infection with conjunctivitis, otitis,

pneumonia, enteritis and infection of the genital
organs

Reptiles Necrotic stomatitis and other necrotic lesions,

especially in captive snakes

Many animal species Infection of burns and other wounds, diarrhoea,

genital and nosocomial infections

P.pseudomallei Many animal species Melioidosis (pseudoglanders)

Horses
The disease can mimic glanders
Cattle
Acute and chronic forms with localisation of lesions in
lungs, joints and uterus
Sheep
Arthritis and lymphangitis predominate
Goats
Loss of condition, respiratory and central nervous
disturbances, arthritis and mastitis
Pigs
As for goats but in addition diarrhoea and abortion

Dogs Febrile disease with localising suppurative foci

P.mallei Horses and other Glanders: acute form with high fever, mucopurulent
equids nasal discharge, respiratory signs, septicaemia and
death within 2 weeks
Chronic forms of glanders
• Pulmonary: small nodules in lungs that break down
and discharge P.mallei into the bronchioles
• Cutaneous form: Farcy, which is a lymphangitis with
ulcers along lymphatic vessels of the limbs and chest.
The ulcers eventually heal leaving 'star shaped' scars.

Acute, septicaemic disease

 98

Humans, cats and

other animals

PASTEURELLA AND MANNHEIMIA

Pasteurella and Mannheimia species are small (0.2 x 1-2 µm) non-motile, Gram-negative rods
or coccobacilli. They are oxidase-positive facultative anaerobes, and most species are catalase-
positive. Although non-enriched media will support their growth, these organisms grow best on media
supplemented with blood or serum. They usually remain viable for only a few days on culture plates.

Some species, such as Mannheimia haemolytica, Pasteurella trehalosi and P. aerogenes can
tolerate the bile salts in MacConkey agar. In smears from infected tissues stained by the Giemsa
method, pasteurellae exhibit bipolar staining.

The family Pasteurellaceae comprises five genera, Actinobacillus, Haemophilus, Mannheimia,

Pasteurella and Lonepinella. These genera share a number of common features and some organisms
have been reclassified within these genera following deoxyribonucleic acid hybridization studies and
16s rRNA sequencing.

The pasteurellae are small (0. 2 µm by up to 2.0 µm), Gram-negative rods or coccobacilli.
They are nonmotile, non-sporing, facultatively anaerobic, fermentative (except for P. anatipesrijer),
oxidase-positive and catalase - positive (except for P. caballi). Although unenriched media support
their growth, they grow best on media supplemented with serum or blood.

Recent Changes in Nomenclature

Previous name Present name

Pasteurella ureae Actinobacillus ureae

Haemophilus avium Pasteurella avium
Pasteurella pneumotropica Pasteurella dogmatis
(Henriksen biotype)

USUAL HABITAT

Pasteurella species are worldwide in distribution with wide spectrum of hosts. Most are
commensals on the mucous membranes of the upper respiratory and intestinal tracts of animals. The
carrier rate for different species varies greatly. Most Pasteurella and Mannheimia species are
commensals on the mucosae of the upper respiratory tract of animals. Their survival in the
 99

environment is relatively short.

Isolation

The routine medium for the isolat ion of Pasteurella spp. is ox or sheep blood agar. Clinical
materials should be inoculated on both blood and MacConkey agars. Selective medium con taining
clindamycin (2 µg / ml) should be used for the isolation of P. mulrocida from porcine nasal swabs.
The plates are in cubated aerobically at 37 °C for 24 48 hours. P. anatipestifer grows best on blood or
serum agar under 5-10 per cent CO2 (a candle-jar is satisfactory).

Colonial morphology

The colonies of all spec ies are usually evident in 24 hours. They are of moderate size, round
and greyish. P.anatipestifer produces small "dewdrop' colonies within 48 hours. The colonies of P.
pneumootropica are non-haemolytic and somewhat similar to those of P. multocida. Type A strains of
P.multocida often produce relatively large, mucoid colonies due to their large capsules of hyaluronic
acid. P. multocida has a characteristic 'sweetish' odour, is non-haemolytic, does not grow on
MacConkey agar and is a good indole producer.

P.haemolytica is beta haemolytic and usually tolerates the bile salts in MacConkey agar to
grow as pinpoint red colonies. It has no odour and does not produce indole. P. testudinis and P.
granulomatis are also haemolytic on blood agar.

Biochemical reactions

Characteristic colonies yielding small, Gram-negative rods or coccobacilli that are oxidase-
positive (the enterobacteria are oxidase-negati ve) and catalase-positive (except for P. cabalii) are
inoculated into TSI slopes. The usual reaction is a yellow slant and butt with no gas or H2S production.
P. anatipestifer is a non-fermenter and may eventually be reclassified in another genus. It is non-hae
molytic, does not g row on MacConkey agar and is indole and urease-negative.

PATHOGENESIS AND PATHOGENICITY

Many P.multocida infections are endogenous. The organisms, which are normally
commensals of the upper respiratory tract, may invade the tissues of immunosuppressed animals.
Exogenous transmission can also occur either by direct contact or through aerosols. Factors of
importance in the development of disease include adhesion of the pasteurellae to the mucosa and the
avoidance of phagocytosis. Fimbriae may enhance mucosal attachment and the capsule, particularly
in type A strains, has a major antiphagocytic role. In septicaemic pasteurellosis, severe endotoxaemia
and disseminated intravascular coagulation cause serious illness which can prove fatal.

Four main virulence factors have been identified in strains of M. haemolytica and P.trehalosi;
fimbriae which may enhance colonization; a capsule that inhibits complement-mediated destruction
of the organisms in serum; endotoxin which can alter bovine leukocyte functions and is directly toxic
to bovine endothelial cells; leukotoxin, a pore-forming cytolysin that affects leukocyte and platelet
functions when present at low concentrations and causes cytolysis at high concentrations.

The subsequent release from damaged cells of lysosomal enzymes and inflammatory
mediators, such as tumour necrosis factor-a and eicosanoids, contributes to severe tissue damage in
these infections.

DIAGNOSTIC PROCEDURES

There may be a history of exposure to stress arising from transportation or overcrowding.

Suitable specimens for laboratory examination from live animals include tracheobronchial aspirates,
 100

nasal swabs or mastitic milk. Tissue or blood smears from septicaemic cases, stained by Giemsa or
Leishman methods, may reveal large numbers of bipolar-staining organisms.

Specimens should be cultured on blood agar and MacConkey agar. Plates are incubated aerobically at
37°C for 24 to 48 hours. Blood agar, supplemented with neomycin, bacitracin and actidione, can be
used for the isolation of P.multocida from heavily contaminated specimens.

Identification criteria for isolates:

- Colonial characteristics

- Growth on MacConkey agar

- Positive oxidase test

- Biochemical profile

- Serological tests are generally of little diagnostic value in the majority of the diseases caused
by pasteurellae and Mannheimia species.

CLINICAL INFECTIONS

Clinical infections caused by pasteurellae and Mannheimia species in domestic animals are
mainly attributable to P.muliocida, M.haemolytica and P.trehalosi. Pasteurella multocida has a wide
host range whereas M.haemolytica is largely restricted to ruminants and P.trehalosi to sheep. The
diseases associated with P.multocida infection include haemorrhagic septicaemia in ruminants and
occasionally in other domestic species, porcine atrophic rhinitis, fowl cholera and bovine pneumonic
pasteurellosis. However, the main aetiological agent of bovine pneumonic pasteurellosis is M.
haemolytica, and this organism is also responsible for pneumonia in sheep and septicaemia in young
lambs. Infection with P.trehalosi frequently results in septicaemia in older lambs.

Mannheimia haemolytica can cause a severe necrotizing mastitis in ewes and both
P.multocida and M.haemolytica has been isolated occasionally from cases of bovine mastitis. Both
organisms have also been implicated aetiologically in the enzootic pneumonia complex of calves.

HAEMORRHAGIC SEPTICAEMIA (HS)

Haemorrhagic septicaemia or barbone is an acute, potentially fatal septicaemia mainly

affecting buffaloes and cattle. Predisposing factors such as overwork, poor body

Buffaloes tend to be more susceptible to the disease than cattle. All ages can be affected but
in endemic areas the disease is most common in animals between 6 and 24 months of age. Older
animals may have a degree of immunity from previous exposure.

Clinical signs

The incubation period of the disease is 2 to 4 days and the course ranges from 2 to 5 days.
Death, without prior signs of illness, may occur within 24 hours of infection. Sudden onset of high
fever, respiratory distress and a characteristic oedema of the laryngeal region are features of the
disease. The oedema may extend to the throat and parotid regions and to the brisket. Recumbency is
followed by death from endotoxaemia. Mortality rates are usually over 50% and can approach 100%.
 101

Diagnosis

A history of acute disease with high mortality in areas where haemorrhagic septicaemia is
endemic may suggest a presumptive diagnosis of the condition. Gross pathological changes may
include widespread petechial haemorrhages, enlarged haemorrhagic lymph nodes and blood-tinged
fluid in the pleural cavity and the pericardial sac. Giemsa-stained blood smears from a recently-dead
animal often reveal large numbers of bipolar-staining organisms.

Isolation, identification and serotyping the P.multocida isolate is confirmatory. Serotypes B:2
and E:2 are the specific strains associated with the disease. An antibody titre of 1:160 or above in an
indirect haemagglutination test is indicative of recent exposure to the pathogen.

Treatment and control

Antibiotic therapy in the early febrile stage is usually effective. Although the organism is
susceptible to penicillin, tetracyclines are more often used. A slaughter policy for affected and in-
contact animals is usually pursued in countries where the disease is exotic. Vaccines available for
control of the disease include bacterins and a modified live vaccine. Latent carriers can be detected
using immunohistochemical techniques on samples of tonsillar tissue.

BOVINE PNEUMONIC PASTEURELLOSIS (SHIPPING FEVER)

Shipping fever, characterized by severe bronchopneumonia and pieurisy occurs most

commonly in young cattle within weeks of being subjected to severe stress, such as transportation,
assembly in feedlots and close confinement. The condition is commonly associated with
M.haemolytica, although P.multocida has also been isolated from lungs of affected cattle.

Clinical signs

Clinical features include sudden onset of fever, depression, anorexia, tachypnoea and serous
nasal discharge. In mixed infections, there is usually a marked cough and ocular discharge. Morbidity
rates can reach 50% and mortality rates range from 1-10%.

Diagnosis

There may be a history of exposure to stress factors and of sudden onset of respiratory
disease. Gross pathological findings are of diagnostic value. Cytospin preparations from
bronchoalveolar lavage usually reveal large numbers of neutrophils. Isolation of M.haemolytica, often
in association with other pathogens, from bronchoalveolar lavage or affected lung tissue is
confirmatory.

Treatment and control

Affected animals must be isolated and treated early in the course of the disease. Treatment
with oxytetracycline, potentiated sulphonamides and ampicillin is usually effective.

Stress factors must be kept to a minimum. Procedures such as castration, dehorning,

branding and anthelmintic therapy should be carried out several weeks before young cattle are
transported. Vaccination regimes for respiratory pathogens should be completed at least 3 weeks
before transportation. Vaccines for M. haemolytica which incorporate modified leukotoxin and
surface antigens may induce protection. Recently, a vaccine containing serotypespecific antigens of
both M. haemolytica A1 and M. haemolytica A6 has been developed.
 102

PASTEURELLOSIS IN SHEEP

Outbreaks of ovine pneumanic pasteurellosis are usually caused by M. haemolytica whereas

P.multocida tends to produce sporadic cases of the disease. Manheimia haemolytica is a commensal
of the upper respiratory tract in a proportion of healthy sheep.

Septicaernic pasteurellosis in lambs less than 3 months of age is caused by M. haemolytica.

In older animals between 5 and 12 months-of-age, septicaemic pasteurellosis is usually associated
with P.trehalosi infection. Pasteurella trehalosi is found in the tonsillar tissues of carrier sheep. As
with most other pasteurella infections, clinical disease may be precipitated by a range of predisposing
factors including transportation. Affected sheep which tend to be in good bodily condition may die
suddenly and the mortality rate may approach 5%.

ATROPHIC RHINITIS OF PIGS

Toxigenic strains of P.multocida type D or A cause a severe, progressive form of atrophic

rhinitis. These toxigenic P.multocida isolates are designated AR+ (atrophic rhinitis-positive) strains.
Infection with Bordetella bronchiseptica may cause mild, non-progressive turbinate atrophy without
significant distortion of the snout.

Clinical signs

Early signs, usually encountered in pigs between 3 and 8 weeks of age, include excessive
lacrimation, sneezing and, occasionally, epistaxis. The snout gradually becomes shortened and
wrinkled. As the disease progresses, a distinct lateral deviation of the snout may develop. Atrophic
rhinitis is rarely fatal. Affected pigs are usually underweight and damage to the turbinate bones may
predispose to secondary bacterial infections of the lower respiratory tract.

Diagnosis

In severely affected pigs characteristic facial deformities are diagnostic. Visual assessment
of the extent of turbinate atrophy can be made following slaughter by transverse section of snouts
between the first and second premolar teeth. Isolation and identification of P.multocida should be
followed by tests to confirm that the isolate is a toxigenic strain. Suitable tests include demonstration
of toxicity for tissue culture cells, an ELISA test for toxin detection and the detection of the toxin gene
by a polymerase chain reaction technique.

Control

Chemoprophylaxis with sulphonamides, trimethoprim, tylosin or tetracyclines in weaner,

grower and sow rations could be considered. Improvement in husbandry must be instituted to
minimize the influence of predisposing factors. Vaccination with a combined B.brunchiseptica
bacterin and P. multocida toxoid may reduce the severity of the disease and improve growth rates.
Sows should be vaccinated at 4 and 2 weeks before farrowing and young piglets at 1 week and 4
weeks of age.
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FOWL CHOLERA

Fowl cholera is a primary avian pasteurellosis caused by P.multocida capsular type A. It is

highly contagious and affects both domestic and wild birds. The disease usually presents as an acute
septicaemia which is often fatal. Turkeys tend to be more susceptible than chickens.

Post mortem lesions include haemorrhages on serous surfaces and accumulation of fluid in
body cavities. In sporadic chronic cases of the disease, the signs and lesions are often related to
localized infections. The wattles, sternal bursae and joints are usually swollen due to the
accumulation of fibrinopurulent exudates.

In the acute septicaemic form of the disease, numerous characteristic bipolar-staining

organisms can be detected in blood smears and P.multocida can be isolated from blood, bone
marrow, liver or spleen. The bacterium may be difficult to isolate from chronic lesions.

Medication of the feed or water with sulphonamides or tetracyclines early in an outbreak of

acute disease may decrease the mortality rate. Polyvalent adjuvant bacterins are widely used.
Autogenous vaccines may be required if the commercial vaccines are ineffective. Modified live
vaccines are available in some countries.

SNUFFLES IN RABBITS

Snuffles is a common, recurring, purulent rhinitis in rabbits which is caused by type A strains
of P.multocida. Bordetella bronchiseptica infection may sometimes cause similar clinical signs.
Pasteurella multocida is a commensal in the upper respiratory tract of healthy carrier rabbits.

Clinical disease is often precipitated by stress factors such as overcrowding, chilling,

transportation, concurrent infections and poor ventilation resulting in high levels of atmospheric
ammonia. There is a purulent nasal discharge which cakes on the forelegs because affected rabbits
paw their noses. Sneezing and coughing may be observed. Sequelae include conjunctivitis, otitis
media and subcutaneous abscessation. Bronchopneumonia may develop in young rabbits.

Treatment or prophylactic therapy with antibiotics may be of value. Predisposing stress

factors must he eliminated. No vaccine is available.

Principal hosts and diseases of the Pasturella species

Species Principal host(s) Disease

P. multocida

Type A Cattle Part of 'shipping fever' complex

Part of the 'enzootic pneumonia' complex in calves
Occasional but severe mastitis

Pleuropneumonia
Sheep Mastitis

Pneumonia (often secondary)

Pigs

One cause of 'snuffles', pleuropneumonia,

 104

Rabbits abscesses, otitis media, conjunctivitis and genital

infections

Fowl cholera (primary infection)

Poultry

Pneumonia and other infections in stressed

Many domestic and animals
wild animals Commensals in respiratory and digestive tract

Type B Cattle, water buffalo, Epizootic haemorrhagic septicaemia (primary

bison , yak and other infection)
ruminants Nasopharynx of carrier animals
South-East Asia and other countries

Type D Pigs Atrophic rhinitis (with or without Bordetella

bronchiseptica )

Pigs, less commonly Pneumonia (usually secondary

other domestic
animals
and poultry

Type E Cattle and water Epizootic haemorrhagic septicaemia (primary

buffalo infection) Africa only

Type F Turkeys mainly Role in disease not clear

P. haemolytica

Biotype A Cattle Part of 'shipping fever' complex

Pneumonia (primary or secondary)

Sheep Enzootic pneumonia (primary or secondary).

Septicaemia in lambs under 3-months-old.
Gangrenous mastitis ('blue bag')

Biotype T Sheep Septicaemia in lambs 5-12 months-old

P. pneumotropica Rodents Pneumonia (secondary) and abscesses (possibly

from bites)
Commensal in nasopharynx

Dogs and cats Present in the nasopharynx of some animals. Not

a significant pathogen

P.canis Dogs (man) Recovered from the oral cavity of dogs and from
dog bites in humans

P.dagmatis Dogs, cats (man) Present in the oral cavity and intestinal tract of
dogs and cats and recovered from dog and cat
 105

bites in humans

P.stomatis Dogs, cats Isolated from the respiratory tract but not usually
pathogenic

P. caballi Horses Respiratory infections including pneumonia

P. aerogenes Pigs (man) Commensal in intestine of pigs. Rarely pathogenic

but one isolate associated with abortion.

Abscesses from pig bites in humans

P. anatipestifer Ducklings mainly but 'New duck disease': severe fibrinous polyserositis
also chickens, in
turkeys, pheasants, ducklings 1-8 weeks old
water fowl

P. anatis Ducks Recovered from intestines. Pathogenicity not

demonstrated

P. gallinarum Poultry Commensal in respiratory mucosa. Occasional

low grade infections

P. avium Chickens Isolated from the respiratory tract of healthy birds

P. fangaa
P. vofantium Pathogenicity not demonstrated for any of the
three species

P. testudinis Turtles, tortoises Associated with abscesses. Possibly

opportunistic

P. granufomatis Cattle (Brazil) Fibrogranulomatous disease

 106

ACTINOBACILLUS

Actinobacillus species are non-motile, Gram-negative rods (0.3 to 0.5 x 0.6 to 1.4 µm) which
occasionally have a coccobacillary appearance. They are non-motile, non-sporeforming and non-acid-
fast. A surface slime is present in the three major species (A. ligllieresii. A. equuli and A. suis) and
may be related to the stickiness of their colonies on agar. Surface cultures have low viability and die in
5-7 days. The actinobacilli ferment carbohydrates, without the production of gas, within 24 hours and
most species produce urease and grow on MacConkey agar. Most species are urease and oxidase
positive. The reactions in the oxidase and catalase tests are variable. The genus is still in a state of
flux, mainly because of the close similarities between actinobacilli and species in the genera
Pasteurella and Haemophilus.

These facultative anaerobes ferment carbohydrates producing acid but not gas. Actinobacilli
exhibit some host specificity and are mainly pathogens of farm animals.

USUAL HABITAT

Actinobacilli are commensals on mucous membranes of animals particularly in the upper

respiratory tract and oral cavity. As actinobacilli cannot survive for long in the environment, carrier
animals play a major role in transmission.

COLONIAL APPEARANCE

• A. lignieresii: small, glistening colonies develop in 24 hours. They are usually slightly sticky (viscid)
on primary isolation but lose th is characteristic on subculture. The colonies are non-haemolytic and
develop to about 2 mm in diameter in 48 hours. The organism grows well on MacConkey agar, the
colonies are at first pale but become pinkish as A. lignieresii is a late lactose-fermemer.

• A. equuli: some strains are haemolytic and the colonies are sticky with this feature remaining on
subculture. I t is a lactose-fermenter on MacConkey agar.
 107

• A. suis: all strain s are haemolytic with colonies similar to those of A. lignieresii but more sticky. It
grows well on MacConkey agar.

• A.pleuropneumoniae: small (1 mm) colonies surrounded by a zone of beta-haemolysis, which

somewhat resemble those of a beta-haemolytic streptococcus. Two distinct colony forms are
possible, one waxy and the other a soft glistening type. No growth occurs on MacConkey agar.

• A. capsulatus: the cells are capsulated and very sticky colonies are produced on blood agar. It grows
well on MacConkey agar.

• A. actinomycetemcomitans: the colonies are small ( 1 mm after 2-3 days), adherent to the medium
and difficult to break-up. Colonies are described as 'star-like'. No growth occurs on MacConkey agar.

• A. seminis: small, round, pinpoint colonies that are greyish-white, non-haemolytic and appear after
24 48 hours on blood agar. No growth occurs on MacConkey agar.

• A. ureae: colonies on blood agar are mucoid and are usually accompanied by some greening of the
medium.

BIOCHEMICAL REACTIONS

A. pleuropneumoniae enhances the haemolysis caused by the beta-haemolysin of

Staphylococclls aureus in a CAMP test. A. seminis is usually catalase-positive but oxidase- negative,
and almost completely unreactive in conventionally performed biochemical tests. A few strains show
slight acid production, after incubation periods of up to 28 days, in arabinose, fructose, mannose,
trehalose, glucose, maltose, mannitol and xylose.

Most strains are reported as being nitrate, H2S, MR, VP, indole, urease, Phosphatase,
gelatinase and citrate negative.

PATHOGENESIS AND PATHOGENICITY

The virulence factors possessed by the actinobacilli are poorly defined with the exception of
those associated with A. pleuropneumuniae, the cause of pleuropneumonia in pigs.

CLINICAL INFECTIONS

Actinobacilli can cause a variety of infections in farm animals including 'timber (wooden)
tongue' in cattle, pleuropneumonia in pigs and systemic disease in foals and piglets.

ACTINOBACILLOSIS IN CATTLE

Actinobacillosis, a chronic pyogranulomatous inflammation of soft tissues, is most often

manifest clinically in cattle as induration of the tongue, referred to as timber tongue. Potentially
important lesions occur in the oesophageal groove and the retropharyngeal lymph nodes.

The aetiological agent, Acrinobacillus lignieresii is a commensal of the oral cavity and the
intestinal tract. It can survive for up to 5 days in hay or straw. The organisms enter tissues through
erosions or lacerations in the mucosa and skin. A localized pyogranulomatous response is associated
with club colonies containing the bacteria. In addition, spread through the lymphatics to the regional
lymph nodes may induce pyogranulomatous lymphadenitis.

Bovine actinohacillosis is usually a sporadic disease, although herd outbreaks of limited

extent can occur. Animals with timber tongue have difficulty in eating and drooling of saliva.
Involvement of the tissues of the oesophageal groove can lead to intermittent tympany and
enlargement of the retropharyngeal lymph nodes can cause difficulty in swallowing and stertorous
 108

breathing. Lesions of cutaneous actinobacillosis may be found on the head, thorax, flanks and upper
limbs.

Animals with ulcerated discharging lesions can contaminate the environment. Localized
pyogranulomatous lesions in the retropharyngeal lymph nodes are often found at slaughter.

Diagnosis

Induration of the tongue is characteristic of the disease and there may be a history of grazing
rough pasture. Spccimens for laboratory examination include pus, biopsy material and tissues from
lesions at post-mortem. Gram-negative rods are demonstrable in smears from exudates.

Pyogranulomatous foci containing club colonies may be evident in tissue sections. Cultures
on blood agar and MacConkey agar are incubated aerobically at 37°C for 24 to 72 hours.

Isolation

Most of the actinobacilli can be cultured on sheep or ox blood agar and some will grow on
MacConkey agar. However, A. pleuropleuropneumoniae benefits from factor V that can be provided
by chocolate agar or a staphylococcal streak on blood agar. The growth of all the actinobacilli and
particularly A. pleuropleuropneumoniae is improved by 5-10 per cent CO2 (a candle-jar is satisfactory).
The inoculated plates are incubated at 37°C for 24-72 hours.

Identification criteria for isolates:

 Small, sticky, non-haemolytic colonies on blood agar

 Slow lactose fermentation on MacConkey agar

 Biochemical profile

Treatment and Control

Animals with discharging lesions should be isolated. Sodium iodide parenterally or potassium
iodide orally is effective. Potentiated sulphonamides or a combination of penicillin and streptomycin
are usually effective. Oral isoniazid for 30 days has been used in animals with refractory lesions.
Rough feed or pasture which may damage the oral mucosa should he avoided.

INFECTIONS IN OTHER ANIMALS CAUSED BY A. LIGNIERESII

Cutaneous actinobacillosis of sheep presents as granulomatous lesions mainly on the head

without tongue involvement. Granulomatous mastitis in sows, bite wounds in dogs and glossitis in a
horse have been attributed to infection with A. lignieresii.

PLEUROPNEUMONIA OF PIGS

Pleuropneumonia, caused by A. pleuropneumoniae, can affect susceptible pigs of all ages

and occurs in major pig rearing regions worldwide. This highly contagious disease, primarily in pigs
under 6 months of age, appears to be increasing in prevalence as a consequence of intensive rearing
practices.

Pathogenesis and pathogenicity

Virulence factors associated with A.pleuropneumoniae have been partially elucidated.

Virulent strains possess capsules which are both antiphagocytic and immunogenic, whereas non-
 109

encapsulated strains are avirulent. Fimbriae and other adhesins allow the organisms to attach to cells
of the respiratory tract. Actinobacillus pleuropneumoniae produces three related cytotoxins which
belong to the repeats-in structural- toxin (RTX) cytolysin family. These toxins act by producing pores
in cell membranes. Neutrophils chemically attracted to infected lung tissue are damaged and release
lytic enzymes. The sustained inflammatory response is considered to be a major factor in causing
rapid tissue necrosis.

Clinical signs and epidemiology

Subclinical carrier pigs, which are encountered in unaffected populations, harbour the
organisms in the respiratory tract and tonsillar tissues. Poor ventilation and sudden drops in ambient
temperature seem to precipitate disease outbreaks. Aerosol transmission occurs in confined groups.
In outbreaks of acute disease, some pigs may be found dead and others show dyspnoea, pyrexia,
anorexia and a disinclination to move. Blood-stained froth may be present around the nose and mouth,
and many pigs show cyanosis.

Pregnant sows may abort. Morbidity rates can range from 30-50% and case fatality rates may
reach 50%. Concurrent infections with Pasteurella multocida and mycoplasma may exacerbate the
condition. At post-mortem areas of consolidation and necrosis are found in the lungs along with
fibrinous pleurisy. Blood-stained froth may be found in the trachea and bronchi.

Diagnosis

There may be a history of ventilation failure or environmental temperature decrease prior to

an outbreak of pulmonary disease. Specimens for laboratory examination should include tracheal
washings or affected portions of lung tissue. Areas of haemorrhagic consolidation close to the main
bronchi and severe fibrinous pleuritis may suggest this condition.

Specimens, cultured on chocolate agar and blood agar, are incubated in an atmosphere of 5-
10% CO2 at 37°C for 2 to 3 days.

Identification criteria for isolates:

 Small colonics surrounded by clear haemolysis

 No growth on MacConkey agar

 Positive CAMP test with Staphylococcus aureus

 Biochemical profile

 Twelve serotypes and two biotypes are recognised and isolates belonging to biotype 1 require
V factor (NAD) for growth whereas those belonging to biotype 2 are NAD-independent.

 The serotypes prevalent in a particular region should be identified prior to the implementation
of vaccination programmes. Serological techniques are also used for epidemiological studies.

 Immunofluorescent techniques or PCR-based techniques may be used to demonstrate the

organism in tissues.

Treatment

As antibiotic resistance is encountered in some strains, chemotherapy should be based on

the results of antibiotic susceptibility testing. Prophylactic administration of antibiotics to in-contact
pigs may limit the severity of clinical disease.
 110

Control

Polyvalent bacterins may induce protective immunity but fail to prevent transmission or the
development of a carrier state. A subunit vaccine containing toxoids of the three A.
pleuropneumoniae toxins and capsular antigen has been developed. Predisposing factors such as
poor ventilation, chilling and overcrowding should be avoided.

SLEEPY FOAL DISEASE

Sleepy foal disease is an acute, potentially fatal septicaemia of newborn foals caused by
Actinobacillus equuli. Although primarily a pathogen of foals, A.equuli occasionally produces disease
conditions, such as abortion, septicaemia and peritonitis, in adult horses. The organism is found in
the reproductive and intestinal tracts of mares. Foals can be infected inutero and after birth via the
umbilicus. Affected foals are febrile and recumbent. Death usually occurs in 1 to 2 days. Foals which
recover from the acute septicaemic phase may develop polyarthritis, nephritis, enteritis or pneumonia.
Foals dying within 24 hours of birth have petechiation on serosal surfaces and enteritis.
Meningoencephalitis can be detectable histologically. Foals which survive for 1 to 3 days have typical
pin-point suppurative foci in the kidneys.

Diagnosis

 History of the disease occurring on the premises in previous seasons.

 The clinical signs in a neonatal foal may suggest the disease.

 Specimens should be cultured on blood agar and MacConkey agar and incubated aerobically
at 37°C for 1 to 3 days.

 Identification criteria for isolates:

- Sticky colonies with variable haemolysis on blood agar

- Lactose-fermenting colonies on MacConkey agar

- Biochemical profile

Treatment and Control

Unless the disease is detected early, antimicrobial therapy is of little benefit. The organism is
usually susceptible to streptomycin, tetracyclines and ampicillin. Supportive treatment includes blood
transfusion arid bottle-feeding with colostrum. Mares which have had affected foals should be
monitored closely at subsequent foalings. Good hygiene should be observed. Prophylactic antibiotic
therapy may be considered fur newborn foals. No commercial vaccines are available.

DISEASES CAUSED BY THE PATHOGENIC ACTINOBACILLI

Species Host(s) Disease

A. lignieresii Cattle Bovine actinobacillosis: 'wooden (timber) tongue'
and pyogranulomatous lesions around the head,
neck and limbs and occasionally in the internal
organs

Sheep Pyogranulomatous lesions usually in head and neck

region

Pigs Rare granulomatous abscesses in mammary glands

A. equuli Neonatal foals Sleepy foal disease (shigellosis or viscosum
 111

infection):
septicaemia and those foals that survive for a few
days develop purulent nephritis, pneumonia and
arthritis

Postnatal foals Purulent arthritis, enteritis and infection of

aneurysms

Mares Occasional abortion and/or septicaemia

Pigs Arthritis in piglets and endocarditis in older pigs

Occasional infections
Calves, dogs, rats
and rabbits
A. suis Pigs under 3 Septicaemia
months-old

Older pigs Arthritis, pneumonia and pericarditis

Neonatal and Some strains cause syndromes similar to those in

postnatal foals foals associated with A equuli
A. pleuropneumoniae Pigs Acute: severe fibrinous pleuropneumonia.
Chronic: pleuritis and pulmonary sequestration and
abscessation.
Morbidity and mortality high in non-immune pigs
A. capsulatus Rabbits Arthritis
A. actinomycetem Humans Role in periodontal disease and endocarditis
comitans
Rams Associated with epididymitis
A. seminis Rams Epididymitis
A.ureae Humans Upper respiratory tract infections

Sows Reported as a cause of abortion

HAEMOPHILUS
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INTRODUCTION

The genus Haemophilus contains small, nonmotile, nonsporing, oxidase positive, pleomorphic,
gram negative bacilli that are parasitic on human beings or animals. Haemophilus means blood loving
organisms.

Haemophilus species are small (less than 1 pm x 1 to 3 µm), Gram-negative rods, which often
appear coccobacillary and may occasionally form short filaments. These motile organisms, which are
facultative anaerobes with variable reactions in catalase and oxidase tests, do not grow on
MacConkey agar. They are fastidious bacteria requiring one or both of the growth factors X (haemin)
and V (nicotinamide adenine dinucleotide, NAD). Optimal growth occurs in an atmosphere of 5-10%
C02 on chocolate agar which supplies both X and V factors. Small, transparent, dewdrop-like colonies
are formed by most Haemophilus species after incubation for 48 hours.

Colonies of H.somnus have a yellowish hue and some isolates are haemolytic on sheep blood
agar. The main pathogens in the genus are H.somnus in cattle and sheep, H.parasuis in pigs and
H.paragallinarum which is responsible for infectious coryza of chickens.

The haemophili are motile, facultative anaerobes, produce acid from glucose, reduce nitrates
and are variable in the oxidase and catalase tests. They are nutritionally fastidious, will not grow on
MacConkey agar and grow best on chocolate agar (supplying the X and V factors) under 5- 10 per
cent CO2 at 37°C.

The growth of many of the Haemaphilus species is enhanced by 10 per cent CO2, As this is
not inhibitory for any of them, CO2 should be used for routine isolation. The inoculated chocolate agar
plates are incubated under 10 per cent CO2 at 35-37°C for 3-4 days, although some growth may be
seen after 24 hours.

COLONIAL MORPHOLOGY

Small dewdrop-like colonies may appear after 24 48 hours' incubation and none are
consistently haemolytic. A few strains of H. somnus may show a frank clearing around the colonies
especially on Columbia-base sheep blood agar. H. somnus colonies may appear yellowish especially
in a loopful of growth or on a confluent lawn.

Haemophilus piscium: H. piscium, responsible for ’ulcer disease' in trout, is probably not a
true Haemophilus species as it does not require either the X or V factors for growth. It is a Gram-
negative rod (0.5-0.7 x 2-3 µm), non-motile and will not grow at 37°C, the optimum temperature for
growth being 20- 25°C. Media for isolation should be supplemented with a peptic digest of fish tissue.
Colonies develop to 1-3 mm in diameter and are circular, convex and cream-coloured. On blood agar
complete haemolysis is produced.

USUAL HABITAT

Haemophilus species are commensals on the mucous membranes of the upper respiratory
tract. They are susceptible to desiccation and do not survive for long periods away from their hosts.

PATHOGENESIS AND PATHOGENICITY

Young or previously unexposed animals are particularly susceptible to infections by

Haemophilus species. Specific-pathogen-free pigs, which do not harbour H.parasuis as a commensal,
often develop signs of disease on primary exposure to the pathogen. Environmental stress factors
appear to contribute to the development of Haemophilus infections. Although virulence factors have
not been fully identified, endotoxin is thought to play a role in the pathogenesis of infections.
 113

Haemophilus somnus can adhere firmly to several host cell types, including endothelial and
vaginal epithelial cells. The organism is reported to cause degeneration of macrophages and it
suppresses neutrophil function.

Degeneration of vascular endothelial cells and transmural neutrophil infiltration are prominent
findings in thrombotic meningoencephalitis. Certain outer membrane proteins which confer virulence
allow widespread dissemination of the bacteria in the host. Immunity to H.somnus appears to be
predominantly humoral in nature. However, phase variation in the lipopolysaccharide antigen types
may influence survival and persistence in host animals.

DIAGNOSTIC PROCEDURES

Specimens for laboratory examination depend on the clinical condition and type of lesions.
Haemophilus species are fragile and neither refrigeration nor transport media maintain viability.
Ideally, clinical specimens should he frozen in dry ice and delivered to a laboratory within 24 hours of
collection. Either chocolate agar or blood agar inoculated with a streak of S.aureus incubated under 5
- 10% CO2 at 37°C for 2 to 3 days in a moist atmosphere is used for isolation.

Identification criteria for isolates:

- Small, dewdrop-like colonies after 1 to 2 days

- Enhancement of growth by CO2

- Requirement for X and V growth factors

- Biochemical profile

- Although serological tests have been developed for epidemiological purposes, these tests are
of little diagnostic value because Haemophilus species are widely distributed in animal
populations.

CLINICAL INFECTIONS

The Haemophilus species which are pathogenic for animals tend to be host-specific. Some
Haemophilus species of uncertain pathogenicity which are occasionally isolated from domestic
animals.

INFECTIONS CAUSED BY H. SOMNUS IN CATTLE

Haemophilus somnus is part of the normal bacterial flora of the male and female bovine
genital tracts. The organism can also colonize the upper respiratory tract. Environmental stress
factors contribute to the development of clinical disease. Haemophilus somnus is more resistant in
the environment than most other Haemophilus species. It can survive in nasal discharges and blood
for up to 70 days at ambient temperatures and for up to 5 days in vaginal discharges. Transmission is
by direct contact or by aerosols. Serological surveys indicate that at least 25% of cattle have
antibodies to H.somnus.

Clinical signs

Because septicaemia is commonly associated with H.somnus infection, many organ systems
may be involved and the resulting clinical presentation can be variable. Thrombotic
meningoencephalitis (TME), a common consequence of septicaemia, is encountered sporadically in
 114

young cattle recently introduced to feedlots. Some animals may be found dead and others may
present with high fever and depression, sometimes accompanied by blindness, lameness and ataxia.
Sudden death due to myocarditis has also been described. Arthritis often develops in animals which
survive the acute phase of the disease.

Haemophilus somnus is one of the bacterial pathogens commonly isolated from the enzootic
calf pneumonia complex. Sporadic cases of abortion, endometritis, otitis and mastitis caused by
H.somnus have been recorded.

Diagnosis

Severe neurological signs in young feedlot cattle may be indicative of TME. Multiple foci of
haemorrhagic necrosis, detectable grossly in affected brains at postmortem are consistent with TME.
Vasculitis, thrombosis and haemorrhage are detectable histologically in brain, heart and other of the
male and female bovine genital tracts and other parenchymatous organs.

Treatment and control

Animals with clinical signs of septicaemia should be isolated and those at risk should be
monitored closely to detect early signs of the disease. Although oxytetracycline is usually used for
therapy, penicillin, erythromycin and potentiated sulphonamides are also effective. Commercially-
available bacterins may reduce morbidity and mortality rates if administered one month before
outbreaks of disease is anticipated.

INFECTIONS CAUSED BY HAEMOPHILUS SOMNUS IN SHEEP

Healthy sheep may carry H.somnus in the prepuce or vagina. Epididymitis in young rams,
caused by H.somnus has been recorded. Vulvitis, mastitis and reduced reproductive performance in
ewes have been attributed to infection with H.somnus. The organism has also been associated with
septicaemia, arthritis, meningitis and penumonia in lambs.

INFECTIONS CAUSED BY HAEMOPHILUS SOMNUS IN PIGS

GLASSER'S DISEASE

Glasser's disease, caused by H.parasuis, manifested as polyserositis and leptomeningitis

usually affecting pigs from weaning up to 12 weeks of age. Some cases present as polyarthritis.

Haemophilus parasuis is part of the normal flora of the upper respiratory tract of pigs. Piglets
acquire the organism from sows shortly after birth either by direct contact or through aerosols. The
presence of maternally derived antibodies prevents the development of clinical signs. However,
Glasser's disease may occur sporadically in 2 to 4 week old piglets subjected to stressful
environmental conditions. Active immunity to H. parasuis is usually established by 7 to 8 weeks of
age.

Clinical signs

The incubation period is 1 to 5 days. Clinical signs usually develop in conventionally-reared

pigs 2 to 7 days following exposure to stress factors such as weaning or transportation. Anorexia,
pyrexia, lameness, recumbency and convulsions are features of the disease. Cyanosis and thickening
of the pinnae are often encountered. Pigs may die suddenly without showing signs of illness.

Diagnosis

Because organisms such as Streptococcus suis and Mycoplasma hyorhinis produce

clinicopathological changes similar to those of Glasser's disease, diagnosis requires isolation and
 115

identification of H.parasuis. Postmortem findings in Glasser's disease may include fibrinous

polyserositis, polyarthritis and meningitis.

Isolation and identification of H. parasuis from joint fluid, heart blood, cerebrospinal fluid or
post-mortem tissues of a recently-dead pig is confirmatory.

Treatment and control

Antimicrobial drugs such as tetracyclines, penicillins or potentiated sulphonamides,

administered early in the course of the disease, are usually effective. Predisposing stress factors
should be identified and, where possible, eliminated. Commercially-available bacterins or autogenous
bacterins may stimulate protective immunity. Immunity is serotype specific.

INFECTIOUS CORYZA OF CHICKENS

Infectious coryza, caused by H.paragallinarum, affects the upper respiratory tract and
paranasal sinuses of chickens. Its economic importance relates to loss of condition in broilers and
reduced egg production in laying birds. Chronically ill and, occasionally, clinically normal carrier birds
act as reservoirs of infection. Transmission occurs by direct contact, by aerosols or from
contaminated drinking water. Chickens become susceptible at about 4 weeks after hatching and
susceptibility increases with age.

Clinical signs

The mild form of disease manifests as depression, serous nasal discharge and slight facial
swelling. In severe disease, swelling of one or both infraorbital sinuses is marked and oedema of the
surrounding tissues may extend to the wattles. In laying birds, egg production may be severely
affected. A copious, tenacious exudate may be evident at post-mortem in the infraorbital sinuses and
tracheitis, bronchitis and airsacculitis may be present.

Diagnosis

Facial swelling is a characteristic finding. Isolation and identification of H. paragallinarum

from the infraorbital sinuses of several affected birds is confirmatory. Immunoperoxidase staining
can be used to demonstrate H. paragallinarum in the tissues of the nasal passages and sinuses.
Serological tests such as agglutination tests, ELlSA or agar gel immunodiffusion tests are used to
demonstrate antibodies about 2 to 3 weeks after infection and to confirm the presence of H.
paragallinarum in a flock.

Treatment and control

Medication of water and feed with oxytetracycline or erythromycin should be initiated early in
an outbreak of disease. An all-in-all-out management policy should be implemented and replacement
birds should be obtained from coryza free stock. Good management of poultry units minimizes the
risk of infection. Bacterins may be of value in units where the disease recurs. Vaccines should be
administered about 3 weeks before outbreaks of coryza are anticipated.

DISEASES OF THE HAEMOPHILUS SPECIES

Species Host(s) Disease or significance

H. influenzae Humans Variety of diseases ranging from respiratory infections to

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meningitis

H. parainfluenzae Humans Normal flora of upper respiratory tract and implicated in

urethritis

H. somnus Cattle •Infectious thromboembolic meningoencephalitis

(TEME): septicaemia with infarcts in cerebellum
• Respiratory disease: pneumonia and pleurisy, often in
mixed infections with other agents
• Genital infections such as endometritis and abortion. It
can occur in semen and genital tract of bulls

Commensal of genital tract of sheep and reported as a

Sheep cause of epididymitis and orchitis in rams. May also
cause pneumonia, mastitis, polyarthritis, meningitis and
septicaemia

H.parasuis Pigs • Primary agent of Glasser's disease: polyserositis and

meningitis in young pigs
• Arthritis and pneumonia in older pigs
• Secondary invader in swine influenza or in
enzootic pneumonia (Mycoplasma hyopneumoniae)
H.paragallinarum Poultry Infectious coryza of chicken: nasal discharge, sneezing
and oedema of the face. reduction in egg production

Highly susceptible to the infection

Japanese
quail

H.haemoglobinophilus Dogs Commensal of lower genital tract and sometimes causes

cystitis and neonatal infections. Isolated from
balanoposthitis and vaginitis but the role of the
bacterium in these conditions is uncertain

H. aphrophilus Humans Part of the oral flora, found in dental plaque, and can be
an occasional cause of endocarditis

Dogs Recovered from the pharynx of dogs

H.ovis Sheep Rare reports of bronchopneumonia

H. paracuniculus Rabbits Significance not known. Isolated from the intestine of

rabbits with mucoid enteritis

H.influenzaemurium Mice Respiratory infections and conjunctivitis

H. piscium Trout Ulcer disease: ulcerating inflammation of gills and mouth

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BRUCELLA

Brucella species are small (0.6 x 0.6 to 1.5 pm), nonmotile, coccobacillary, Gram-negative
bacteria. As they are not decolourized by 0.5% acetic acid in the modified Ziehl-Neelsen (MZN)
staining technique, they are classed as MZN-positive. In MZN-stained smears of body fluids or tissues,
they characteristically appear as clusters of red coccobacilli. For taxonomic purposes, all Brucella
species should be classified as Brucella melitensis as DNA hybridization studies have shown that the
genus contains only one species.

Brucella species are aerobic, capnophilic and catalase-positive. Apart from B.ovis and
B.neotomae, they are oxidase-positive. All Brucella species are urease-positive except B.ovis. Brucella
ovis and some biotypes of B.abortus require 5 to 10% CO2 for primary isolation. Moreover, the growth
of other Brucella species is enhanced in an atmosphere of CO2. Media enriched with blood or serum is
required for culturing B.abortus biotype 2 and B.ovis. Recently, brucellae have been detected in sea-
mammals.

Smears are made from specimens and stained by the modified Ziehl- Neelsen (MZN) stain.
Brucellae appear as small, red-staining coccobacilli in clumps because of their intracellular growth.

ISOLATION

The brucellae grow well on 5-10 per cent blood agar. However, other than foetal abomasal
contents and colostrum, the specimens are likely to contain many contaminating bacteria and fungi,
so selective media are usually required. The selective media contain a nutritive blood agar base with 5
per cent sterile sero-negative equine or bovine serum and an antibiotic supplement. The antibiotic
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supplement used in selective media for B.ovis usually differs from that for B. abortus. Skirrow agar is
a satisfactory medium for both the Campylobacter fetus subspecies and for brucellae, including the
most fastidious species such as B. abortus biotype 2, B.canis and B.ovis.

COLONIAL MORPHOLOGY

After 3-5 days' incubation on selective serum agar, pinpoint, smooth, glistening, bluish,
translucent colonies appear. As they age the colonies become opaque and about 2-3 mm in diameter.
Strains of B. abortus, B. suis, B. melitensis and B. neotomae are usually in the smooth form when first
isolated. Colonies of rough morphology occur in each of these species on subculture. B. ovis and B.
canis are always in the rough form. The rough colonies are dull, yellowish, opaque and when touched
with an inoculation loop are found to be friable. Brucellae are nonhaemolytic on blood agar.

BIOCHEMICAL TESTS

For routine identification, a few biochemical tests, together with colonial morphology and
staining properties will presumptively identify the isolate as a Brucella sp. In summary, the brucellae
are non-motile, catalase-positive, oxidase-positive (except B. ovis and B. neotomae), give rapid urease
activity (except B. ovis and some strains of B. melitensis), reduce nitrate and are indole-negative.
Using known positive antisera a rapid slide agglutination test can be used for B.abortus.

USUAL HABITAT

As a general rule, brucellae have a predilection for both female and male reproductive organs
in sexually mature animals and each Brucella species tends to infect a particular animal species.
Infected animals serve as reservoirs of infection which often persists indefinitely. Organisms, shed by
infected animals, can remain viable in a moist environment for many months.

PATHOGENESIS AND PATHOGENICITY

The establishment and outcome of infection with brucellae depend on the number of
infecting organisms and their virulence and also on host susceptibility. Brucellae, which lack the
major outer-membrane lipopolysaccharide, produce rough colonies and are less virulent than those
derived from smooth colonies. Although smooth and rough organisms can enter host cells. Rough
forms are usuallv eliminated unlike smooth forms which may persist and multiply. Virulent brucellae,
when engulfed by phagocytes on mucous membranes, are transported to regional lymph nodes.
Brucellae persist within macrophages but not within neutrophils. Inhibition of phagosome-lysosome
function is a major mechanism for intracellular survival and an important determinant of bacterial
virulence.

However, many of the mechanisms used by brucellae to survive within macrophages are not
fully elucidated. Various stress proteins are thought to allow the organisms to adapt to harsh
conditions encountered within macrophages. In addition, superoxide dismutase and catalase
production may play a role in resistance to oxidative killing. Intermittent bacteraemia results in spread
and localization in the reproductive organs and associated glands in sexually mature animals.
Erythritol, a polyhydric alcohol which acts as a growth factor for brucellae, is present in high
concentrations in the placentae of cattle, sheep, goats and pigs. This growth factor is also found in
other organs such as the mammary gland and epididymis, which are targets for brucellae. In chronic
brucellosis, organisms may localize in joints or inter vertebral discs.

DIAGNOSTIC PROCEDURES

The diagnosis of brucellosis depends on serological testing and on the isolation and
identification of the infecting Brucella species. Care should be taken during collection and
transportation of specimens, which should be processed in a biohazard cabinet. Specimens for
 119

laboratory examination should relate to the specific clinical condition encountered. MZN-stained
smears from specimens, particularly cotyledons, foetal abomasal contents and uterine discharges,
often reveal characteristic MZN-positive coccobacilli. In specimens containing cells, the organisms
appear in clusters.

A nutritious medium such as Columbia agar, supplemented with 5% serum and appropriate
antimicrobial agents, is used for isolation. Plates are incubated at 37°C in 5 to 10% CO2 for up to 5
days. Although CO2 is a specific requirement for individual species, the majority of brucellae are
capnophilic.

Serological testing is used for international trade and for identifying infected herds or flocks
and individual animals in national eradication schemes. Brucellae share antigens with some Gram-
negative bacteria such as Yersinia enterocolitica serotype 0:9, and consequently cross-reactions can
occur in agglutination tests. The polymerase chain reaction can be used to detect brucellae in tissues.

CLINICAL INFECTIONS

Although each Brucella species has its own natural host, B.abortus, B.melitensis and biotypes
of B.suis can infect animals other than their preferred hosts.

BOVINE BRUCELLOSIS

Bovine brucellosis, caused by B.abortus and formerly worldwide in distribution, has been
eradicated or reduced to a low prevalence in many countries through national eradication
programmes. Although acquired most often by ingestion, infection can occasionally follow venereal
contact, penetration through skin abrasions, inhalation or transplacental transmission. Abortion
storms may be encountered in herds with a high percentage of susceptible pregnant cows.

Abortion usually occurs after the fifth month of gestation and subsequent pregnancies are
usually carried to term. Large numbers of brucellae are excreted in foetal fluids for about 2 to 4 weeks
following an abortion and at subsequent parturitions, although infected calves appear normal.
Infection in calves is of limited duration in contrast to cows in which infection of the mammary glands
and associated lymph nodes persists for many years. Brucellae may be excreted intermittently in milk
for a number of years. In bulls, the structures targeted include seminal vesicles, ampullae, testicles
and epididymides. In tropical countries, hygromas involving the limb joints are often observed when
the disease is endemic in a herd.

In affected herds, brucellosis can result in decreased fertility, reduced milk production,
abortions in susceptible replacement animals and testicular degeneration in bulls. Abortion is a
consequence of placentitis involving both cotyledons and intercotyledonary tissues. In the bull,
necrotizing orchitis occasionally results in localized fibrotic lesions.

Diagnosis

Clinical signs are not specific although abortions in first-calf heifers and replacement animals
may suggest the presence of the disease.

Clusters of MZN-positive coccobacilli may be evident in smears of cotyledons and MZN-

positive organisms may also be detected in foetal abomasal contents and uterine discharges.

Isolation and identification of B. abortus is confirmatory.

Identification criteria for isolates:

 Colonial appearance
 120

 MZN-positive organisms

 Bacterial cell agglutination with a high-titered antiserum

 Rapid urease activity

 Biotyping

 A range of serological tests, varying in sensitivity and specificity, is available for the
identification of infected animals.

 Brucellin, an extract of B.abortus, has been used for intradermal testing.

 Molecular methods, such as PCR-based techniques, for the detection of brucellae in tissues
and fluids have been described.

Treatment and control

 Treatment of cattle with brucellosis is not practical. Three types of vaccines are used in
cattle, attenuated strain 19 (S19) vaccine, adjuvanted 45120 vaccine and the more recent
RB51 vaccine:

 S19 vaccine is administered to female calves up to 5 months of age. Vaccination of mature

animals leads to persistent antibody titres.

 45120 bacterin although less effective, has been used in some national eradication schemes.
Even when administered to adult animals, this vaccine does not induce persistent antibody
titres.

 RB51 strain is a stable, rough mutant which induces good protection against abortion and
does not result in serological responses detectable in tests used in conventional brucellosis
surveillance programmes.

DISEASES AND PRINCIPAL HOSTS OF THE BRUCELLA SPECIES

Species Host(s) Diseases

CATTLE Abortion and orchitis

Sheep, goats and Sporadic abortion

B. abortus pigs

Horses Associated with bursitis (poll evil and

fistulous withers)

Humans Undulant fever

B. melitensis GOATS, Sheep Abortion

Cattle Occasional abortion and

excretion in milk

Humans Malta fever

B.suis PIGS Abortion, orchitis, arthritis,

spondylitis and herd infertility
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Humans Undulant fever

B. ovis SHEEP Epididymitis in rams and

sporadic abortion in ewes

B.canis DOGS Abortion, epididymitis, discospondylitis and

permanent
infertility in males

Humans Undulant fever

B. neotomae Desert wood rat Non-pathogenic for the wood rat and has
(Neotoma lepida) not been recovered from any other animal
species

VIBRIO AND RELATED ORGANISM

INTRODUCTION

Vibrios are Gram-negative, rigid, curved rods that are actively motile by means of a polar
flagellum. The name ‘Vibrio’ is derived from the characteristic vibratory motility (from vibrare,
meaning to vibrate). They are asporogenous and noncapsulated. Vibrios are present in marine
environments and surface waters worldwide. The most important member of the genus is Vibrio
cholerae, the causative agent of cholera. It was first isolated by Koch (1883) from cholera patients in
Egypt, though it has been observed earlier by Pacini (1884) and others.

NATURAL HABITAT

Vibrio spp. can be present in both freshwater and seawater as well as in the alimentary tracts
of animals and man.

At least five Vibrio spp. are human pathogens including V. cholerae, the cholera bacillus, and
V.parahaemolyticus which causes food poisoning. Only V. metschnikovii is associated with disease in
domestic animals. It causes a cholera-like disease in chickens and other birds but its geographical
distribution is very limited.

V. anguillarum causes infections in many species of fish especially in salt or brackish water.
It causes high mortality in salt water eels.

VIBRIO CHOLERAE

MORPHOLOGY

The cholera vibrio is a short, curved, cylindrical rod, about 1.5 × 0.2-0.4 mm in size, with
rounded or slightly pointed ends. The cell is typically comma shaped (hence the old name V comma)
but the curvature is often lost on subculture. S-shaped or spiral form may be seen due to two or more
cells lying end to end. Pleomorphism is frequent in old cultures. In stained films of mucus flakes from
acute cholera cases, the vibrios are seen arranged in parallel rows, described by Koch as the ‘fish in
stream’ appearance. It is actively motile, with a single sheathed polar flagellum. The motility is of the
darting type, and when acute cholera stool or a young culture is examined under the microscope, the
actively motile vibrios suggest a ‘swarm of gnats’. The vibrios stain readily with aniline dyes and are
Gram negative and non-acid fast.
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CULTURE CHARACTERISTICS

The cholera vibrio is strongly aerobic, growth being scanty and slow anaerobically. It grows
o o
within a temperature range of 16-40 C (optimum 37 C), Growth is better in an alkaline medium, the
range of pH being 6.4-9.6 (optimum 8.2). NaCl (0.5-1%) is required for optimal growth though high
concentration (6% and above) are inhibitory. It grows well on ordinary media. On MacConkey’s agar,
the colonies are colourless at first but become reddish on prolonged incubation due to the late
fermentation of lactose. On blood agar, colonies are initially surrounded by a zone of greening, which
later become clear due to haemodigestion. In peptone water, growth occurs in about six hours as a
fine surface pellicle, which on shaking breaks up into membranous pieces.

V.metschnikovii: smooth, transparent colonies that are 2-4 mm in diameter at 48 hours. It

may be haemolytic on blood agar and grows poorly on MacConkey agar.

V.anguillarum: small, smooth colonies within 48 hours. It is haemolytic on blood agar.

V.parahaemolyticus: moderate-sized (about 2 mm diameter) colonies in 24 hours, non-

haemolytic on sheep blood agar. It forms greenish colonies on TCBS agar (BBL) and ferments
mannitol causing an acid reaction (yellow) on mannitol salt agar.

A number of special media have been employed for the cultivation of cholera vibrios. They
may be classified as follows:

Holding or transport media

1. Venkatraman – Ramakrishnan (VR) medium: In this medium vibrios do not multiply but remain
viable for several weeks.

2. Cary-Blair medium: It is a suitable transport medium for Salmonella and Shigella as well as for
vibrios.

3. V. anguillarum: blood agar with nutrient agar base and 2.0 per cent NaCl at 20°C for 48 hours.

4. V. merschnikovii and V. parahaemoLyticus: nutrient agar or blood agar at 37°C for 24 48 hours.

Enrichment media

1. Alkaline peptone water at a pH of 8.6

2. Monsur’s taurocholate tellurite peptone water at pH 9.2

Both these are good transport as well as enrichment media.

Plating media

1. Alkaline bile salt agar (BSA) at pH 8.2: The colonies after overnight growth, colonies are moist,
translucent, round discs, about 1-2mm in diameter; with a bluish tinge in transmitted light.

2. TCBS medium: This medium, containing thiosulfate, citrate, bile salts and sucrose, is available
commercially and is very widely used at present. Cholera vibrios produce large yellow convex colonies
which may become green on continued incubation.

3. V. parahaemolyticus will grow on TCBS agar (BBL) formulated for V. cholerae and other enteric
vibrios. Being halophilic, it also grows on mannitol salt agar (BBL) containing 7 .5 per cent NaCl
intended for the pathogenic staphylococci.

BIOCHEMICAL REACTIONS
 123

Carbohydrate metabolism is fermentative, producing acid, but no gas. Cholera vibrios ferment
glucose, mannitol, maltose, mannose and sucrose but not inositol, arabinose or lactose, though
lactose may be split very slowly. Indole is formed and nitrates are reduced to nitrites. These two
properties contribute to the ‘cholera red reaction’ which is tested by adding a few drops of
concentrated sulphuric acid to a 24-hour peptone water culture.

With cholera vibrios, a reddish pink colour develops due to the formation of nitroso-indole,
Catalase and oxidase testes are positive. Methyl red and urease tests are negative. Vibrios
decarboxylate lysine and ornithine but not utilize arginine.

V. cholerae and V.mimicus have only a slight requirement for Na+ (NaCI), but most of the
halophilic Vibrio spp. requires the supplementation of biochemical tests with 1 per cent NaCl.
V.parahaemolyticus belongs to the lysine-decarboxylase-positive, arginine-dihydrolase- negative
group of Vibrio spp. It is distinguished from other members of the group by negative reactions for
sucrose, salicin and cellobiose fennentation but a positive reaction for arabinose. V.metschnikovii is
the only clinically significant Vibrio sp. that is oxidase negative and nitrate-reductase-negative.

RESISTANCE

Cholera vibrios are susceptible to heat, drying and acids, but resist high alkalinity. They are
destroyed at 55°C in 15 minutes. Dried on linen or thread, they survive for 1-3 days but die in about
three hours on cover slips. Survival in water is influenced by its pH temperature, salinity, presence of
organic pollution and other facts.

CLASSIFICATION

1. Serological Classification:

VIBRIOS are classified according to biochemical properties & H antigen into Group A and Group B.

 Group A: This group includes vibrio cholarae and have similar biochemical properties and
Common H (flagellar) Antigen. Group A is again classified based on somatic O antigen into
O1 & Non-O1

 O1 (agglutinable vibrio): These vibrios agglutinate with O1 anitserum.

 (ii) Non-O1 (non-agglutinable vibrio) : This are not agglutinated by O1 antiserum. They are
currently upto O-139.

 O1 are classified into Biotypes-CLASSICAL and ELTOR and these are further classified into
serotypes- OGAWA, INABA, HIKOJIMA

 Subgroup O1 can further be classified into two groups as follows;

Characteristics Classical Eltor

Hemolysis - +
Voges Proskauer test - +
Chick erythrocyte agglutination - +
Polymyxin B Sensitivity + -
Group IV phage susceptibility + -
Group V phage susceptibility - +

2. According to growth requirement

 Genus Vibrio includes 33 species the important are:

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 Non halophillic: It includes following & they are able to grow in media without salts. Eg. V.
cholera

 Halophillic: They are unable to grow in media without salts – have a high requirement of NaCl.

Eg.

V. Parahemolyticus is responsible for food poisoning

V. alginoltyticus is responsible for diarrheal disease

V. vulnificus is responsible for diarrheal disease

PATHOGENECITY OF VIBRIO CHOLERAE

Cholera is an acute diarrheal disease caused by V.cholerae. In its most severe form, cholera
is a dramatic and terrifying illness in which profuse painless watery diarrhea and copious effortless
vomiting may lead to hypovolemic shock and death in less than 24 hours. In treated cases, the
disease may last 4-6 days, during which period the patient may pass a total volume a liquid stool
equal to twice his body weight.

CLINICAL FEATURE OF CHOLERA

 All the clinical features of severe cholera result from this massive loss of fluid and
electrolytes.

 The cholera stool is typically a colourless watery fluid with flecks of mucus, said to resemble
water in which rice has been washed (hence called ‘rice water stools).

 The clinical severity of cholera varies widely, from the rapidly fatal disease to a transient
asymptomatic colonization of the intestine by the vibrios.

 The incidence of mild and asymptomatic infections is more with ELTOR vibrios than with the
classical cholera vibrios.

 The incubation period varies from less than 24 hours to about five days. The clinical illness
may begin slowly with mild diarrhea and vomiting in 1-3 days or abruptly with sudden massive
diarrhea.

PATHOGENESIS

 Vibrio Cholerae enters through orally contaminated food & water.

 If gastric acidity is less then they will be destroyed otherwise they passes to intestine
attaches the mucus membrane breach the epithelial cell & attach to produce toxin.

Cholera Toxin

 It is very similarly to heat labile toxin of E.coli in structural, biochemical, biological & antigenic
properties but is more potent.

 It’s production is determined by filamentous phage integrated with bacterial chromosome.

Mode of action

 Cholera Toxin has 2 sub units Sub-Unit A and Sub-Unit B

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 Sub-Unit B binds to GM1 ganglioside on gut epithelial cells.

 This binding activates A subunit & it divides into A1 & A2.

 A1 activates adenly cyclase wich converts ATP to CAMP.

 CAMP increases the outflow of electrolytes & water to gut lumen & causes diarrhea.

 Sub-Unit A2 act as binding agent for A1 to B

LABORATORY DIAGNOSIS

Specimen

1. Stool

 Collected in acute stage of the disease before administration of Antibiotics.

 Collected by introducing lubricated catheter into rectum or directly in screw capped bottle.

2. Rectal Swab

 Very useful in convalescent carrier in which there is no watery diarrhea.

 Transport Media for Cholera are as follows If chances of delay occur then use V-R medium,
Bile peptone transport medium, Monsur’s medium, Carry Blair medium, Autoclaved sea water

Methods

The Methods for demonstration of the disease are as follows:

1. Direct Demonstration of bacilli

2. Microscopy: For rapid diagnosis, the characteristic darting motility of vibrios and its inhibition by
antiserum can be demonstrated hanging drop method using cholera stool from acute cases or more
reliably after enrichment for 6 hours.

3. Culture Media

 Enrichment media – Alkaline peptone water

 For the plating media are – Bile Salt Agar, & TCBS

Method

If the laboratory facilities are available then the specimen which is collected is directly
inoculated on plating media otherwise it is transported by holding media then inoculated on
enrichment media for 6-8 hrs. Then subculture on plating media after colonies appear the slide
agglutination test with O1 antiserum is done after the species is biochemically identified.

4. Biochemical Reactions

V. Cholera is identified from other by Biochemical reaction as follows it shows Oxidase

positive, Catalese Positive, Indole Positive, V.P. Positive test & Citrate Positive but it shows Urease &
Methyl red test negative.
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5. For isolation of carrier

 More than one cycle of enrichment is required.

 As the excretion of bacilli is intermittent repeated stool examination is required.

Prophylaxis

 Provision of protected water supply and improvement of environmental sanitation

 An ideal cholera vaccine is yet to be found.

DISEASES AND HOSTS OF AEROMONAS, PLESIOMONAS AND VIBRIO SPECIES

Species Host(s) Disease

Vibrio parahaemolyticus Humans Food poisoning, associated with seafoods

V. metschnikovii Chickens and other Cholera-like enteric disease

Birds

V. anguillarum Salt·water eels and Skin necrosis, red areas on skin and
other fish generalisation with high mortality
 127

CAMPYLOBACTER

Campylobacter species are slender, curved motile Gram negative rods (0.2 to 0.5 pm wide)
with polar flagella. Daughter cells which remain joined have a characteristic gull-winged appearance
and long spirals formed by joined cells also occur. These microaerophilic organisms grow best on
enriched media in an atmosphere of increased CO2 and decreased oxygen tension. Many
Campylobacter species grow on MacConkey agar. They are non-fermentative and oxidase-positive
and have variable catalase reactions.

USUAL HABITAT

Many Campylobacter species are commensals in the intestinal tracts of warm-blooded

animals. Campylobacter jejuni and C. lari (formerly C. laridis) colonize the intestines of birds, which
can result in faecal contamination of water courses and stored food. A number of Campylobacter
species are excreted in the faeces of pigs. Campylobacter fetus subspecies venerealis appears to be
adapted principally to bovine preputial mucosa.

DIFFERENTIATION OF CAMPYLOBACTER SPECIES

Campylobacter species are strictly microaerophilic, requiring an atmosphere of 5 to 10%

oxygen and 1 to 10% CO2 for growth. A selective enriched medium such as Skirrow agar is usually
used for primary isolation. Differentiation of isolates is based on colonial morphology and certain
cultural, biochemical and antibiotic-susceptibility characteristics.

COLONIAL MORPHOLOGY

 Campylobacter fetus subspecies venerealis and C.fetus subspecies fetus have small, round,
smooth, translucent colonies with a dewdrop appearance.

 Campylobacter jejuni produces small, flat, grey colonies with a spreading, watery appearance.

 Other Campylobacler species vary somewhat in colonial appearance. C. coli produces a pink-
tan pigment and the growth of C. mucosalis may appear as a dirty-yellow colour on the
inoculating loop.

 Colonies of some Campylobacter species, which may contaminate clinical specimens, can be
slightly pigmented.

 Because Campylobacter species do not ferment carbohydrates, other metabolic activities of

these organisms must be used for identification.

BIOCHEMICAL AND OTHER TESTS

Susceptibility or resistance to nalidixic acid or cephalothin, hydrogen sulphide production,

nitrate reduction, growth at 25°C or 45°C and the catalase reaction are some of the criteria on which a
 128

definitive identification of the Campylobaeter species is based.

C. jejuni is the only species that hydrolyses sodium hippurate. For this test a large loopful of a
24 48 hour culture of C. jejuni is emulsified in 0.4 ml of a 1 per cent aqueous solution of sodium
hippurate and incubated at 37°C for 2 hours. Then 0.2 ml of a ninhydrin solution is added to the tubes
at 37°C. A positive reaction is given by a deep purple colour developing after 10 minutes. The
ninhydrin solution is prepared by adding 3.5 g of ninhydrin to 100 ml of a 1:1 mixture of acetone and
butanol.

PATHOGENESIS AND PATHOGENICITY

Campylobacter fetus subspecies venerealis and C.fetus subspecies fetus are structurally
unusual in that they possess a microcapsule or S layer, which consists of high molecular weight
proteins arranged in a lattice formation. This S layer confers resistance to serum-mediated
destruction and phagocytosis and enhances survival in the genital tract. Virulence factors attributed
to C.jejuni include soluble components with enterotoxin-like activity and poorly defined mechanisms
for attaching to and invading host enterocytes. The contribution of heat stable endotoxin to the
pathogenesis of campylobacteriosis is uncertain.

C. jejuni produces an adhesion, a cytotoxin and a heat-labile toxin similar to that of

Escherichia coli. Transmission of many of the Campyloabacter species, including C. fetus subsp.
fetus, is by the faecal-oral route. C. fetus subsp. vellerealis is transmitted by coitus and infection of
the female genital tract may lead to metritis with resulting death and resorption of the embryo
(infertility) or occasionally to abortion.

DIAGNOSTIC PROCEDURES

Details of the diagnostic methods for individual clinical conditions are presented in relevant
sections. Irrespective of the source of specimens for bacterial isolation, certain general principles
relating to culture techniques apply. Campylobacter species require microaerophilic conditions for
growth, usually supplied by commercially-available generator envelopes which deliver 6% oxygen, 10%
carbon dioxide and 84% nitrogen. Although most pathogenic species grow optimally at 37'C, C.jejuni
requires up to 5 days at 42°C for optimum growth.

Smears from cultures and from clinical specimens should be stained with dilute carbol
fuchsin (DCF) for 4 minutes. This method stains the organisms more intensely than the Gram method.

Identification criteria for isolates:

- Growth only under rnicroaerophilic conditions

- Colonial morphology

- Cell morphology in smears stained with DCF or by immunofluorescence

- Metabolic characteristics and antibiotic susceptibility pattern.

CLINICAL INFECTIONS

The most important consequences of infections with organisms in this group are infertility in
cattle due to C.fetus subspecies venerealis and abortion in ewes caused either by C.fetus subspecies
fetus or by C.Jejuni.

BOVINE GENITAL CAMPYLOBACTERIOSIS

Campylobacter fetus subspecies venerealis, the principal cause of bovine genital

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campylobacteriosis, is transmitted during coitus to susceptible cows by asymptomatic carrier bulls.

The bacteria survive in the glandular crypts of the prepuce and bulls may remain infected indefinitely.
The disease is characterized by temporary infertility associated with early embryonic death; return lo
oestrus at irregular periods and, occasionally, by sporadic abortion. About one-third of infected cows
become carriers.

Campylobacter fetus subspecies venerealis persists in the vagina of carrier cows, a feature
attributed to antigenic shifts in the immunodominant antigens of the S layer proteins. Extension of
infection to the uterus with the development of endometritis and salpingitis can occur during the
progestational phase of the oestrus cycle. Campylobacter fetus subspecies fetus, an enteric
organism acquired by ingestion, can cause sporadic abortions in cows.

Diagnosis

Investigation of the breeding records and vaccination history of an affected herd may suggest
campylobacteriosis. Campylobacter species can be detected by the fluorescent antibody technique in
sheath washings from bulls or cervicovaginal mucus from cows. Isolation and identification of
C.fetus subspecies venerealis from preputial or vaginal mucus is confirmatory.

Vaginal mucus agglutination test detects about 50% of infected, infertile cows on a herd basis.
An ELISA can be used to demonstrate IgA antibodies in vaginal mucus after an abortion. A
polymerase chain reaction has been developed as a rapid screening test for the detection of C. fetus
subspecies venerealis in bull's semen.

Treatment and control

Dihydrostreptomycin, administered either systemically or topically into the prepuce, is used

for treating bulls. Intrauterine administration of dihydrostreptomycin can be used therapeutically.
Vaccination with bacterins in an oil emulsion adjuvant is used therapeutically and prophylactically in
problem herds.

PATHOGENIC AND NONPATHOGENIC CAMPYLOBACTER SPECIES

Species Principal host(s) Disease

C. fetus subsp. Cattle Bovine genital campylobacteriasis (epizootic bovine

venerealis infertility): infertility, early embryonic death and
occasional abortion.
Prepuce of asymptomatic bulls

C. fetus subsp. Sheep Ovine genital campylobacteriosis: outbreaks of

fetus abortion

Cattle Occasional abortions

Man Occasional infections

Cattle, sheep Commensal in the intestinal tract

C. jejuni Sheep Outbreaks of abortion

Dogs, cats, other Enteritis with diarrhoea

animals
and man
 130

Poultry Avian vibrionic hepatitis

Many domestic and wild Commensal in the intestinal tract

animals and birds

C. mucosalis Pigs Associated with the porcine intestinal adenomatosis

complex. Present in intestinal tract of normal pigs

C. hyointestinafis Pigs Associated with the porcine intestinal adenomatosis

complex. Commensal in the intestine of normal pigs

C. coli Pigs, man May cause mild diarrhoea in pigs and enteritis in
man. Commensal in the intestine of pigs. Tends
toNincrease in numbers in pigs with swine dysentery
caused by Serpulina hyodysenteriae

C. cryaerophila Cattle, pigs, sheep, Isolated from faeces of normal animals and
horses infrequently from aborted foetuses. Significance
Unknown

C. laridis Dogs, horses, birds Isolated from faeces. Disease status uncertain

C. sputorum biovar Man Commensal in oral cavity. Considered

sputorum nonpathogenic

C. spulorum biovar Cattle Commensal in preputial cavity of bulls and genital

bubulus tract of cows. Considered to be non-pathogenic

C. sputorum biovar Sheep, cattle Present in the intestinal tract and has been isolated
fecalis from semen and vagina of cattle. Considered to be
Non-pathogenic

C. upsaliensis Dogs, man Isolated from diarrhoeic and normal individuals.

Disease status uncertain

BORDETELLA

The bordetellae are small (0.2- 0.5 x 0.5- 1.0 µm), Gram-negative rods that tend to be
coccobacillary. They are strict aerobes and do not attack carbohydrates but derive energy by the
oxidation of amino acids. B. avium and B. bronchiseptica are motile by peritricholls flagella but B.
 131

pertussis and B. parapertussis are non-motile. All are catalase-positive and oxidase-positive. B.
bronchiseptica and B. aium will grow on MacConkey agar.

The genus Bordetella contains four species, B.pertussis, B.parapertussis, B. bronchiseptica

and B. avium. Bordetella pertussis, the type species, and B.parapertussis are human pathogens
associated with whooping cough in children. Bordetella bronchiseptica infects a wide range of animal
species including man, while B.avium is a pathogen of avian species. The bordetellae are occasional
pathogens which have an affinity for ciliated respiratory epithelium.

Bordetella bronchiseptica and B.avium are small (0.2 to 0.5 x 0.5 to 1.5 pm), Gramnegative
rods with a coccobacillary appearance. They are catalase-positive, oxidase-positive aerobes and are
motile peritrichous bacteria. Because they cannot utilize carbohydrates, they derive their energy
mainly from the oxidation of amino acids and have no special growth requirements. They grow on
MacConkey agar.

NATURAL HABITAT

The bordetellae are inhabitants primarily of the upper respiratory tract of healthy and
diseased humans, animals and birds. B. pertussis and B. parapertussis are human pathogens causing
whooping cough and a mild form of whooping cough, respectively. B. bronchiseptica can be present
in the upper respiratory tract of pigs, dogs, cats, rabbits, guinea-pigs, rats, horses and possibly other
animals. B. avium inhabits the respiratory tract of infected poultry, principally turkeys. Mammalian
infections are mainly transmitted by aerosols but in turkeys indirect spread can occur via water and
litter.

CULTURE

The routine med ia used are sheep blood and MacConkey agars. B. avium and B.
hronchiseptica grow well on both media. The plates are incubated aerobically at 37°C for 24-48 hours.
If isolations are to be attempted from specimens containing a large number of bacterial contaminants,
such as nasal swabs, a selective medium is required. The reason is two-fold - to prevent overgrowth
and to maintain the alkaline to neutral conditions for the bordetellae. Even a few fermentative bacteria
on a medium containing carbohydrates can produce sufficient acid to inhibit the Borderella species.

Several selective media have been described, such as MacConkey agar with 1 per cent
glucose and 20 µg/ml furaltadone or blood agar with 20 µg/ml of clindamycin and 4 µg/ml neomycin.

B. avium will grow well on Smith-Baskerville (SB) medium medium, with or without the
antibiotic supplement. The inoculated SB medium is incubated aerobically at 37°C for 48 hours.

COLONIAL MORPHOLOGY

On sheep or horse blood agar B. bronchiseptica forms very small, convex, smooth colonies
with an entire edge after 24 hours. Some strains may be haemolytic. The colonies of B.avium are
similar but are non-haemolytic. Phase modulation occurs in both species and this is thought to be due
to loss of a capsule-like structure on subculture. The virulent, encapsulated phase I colonies are
convex and shiny, those of phase II are larger, circular and convex with a smooth surface and the
avirulent phase III colonies are large, flat and granular with an irregular edge.

The colonies on MacConkey agar are small, pale with a pinkish hue and amber discolouration
of the underlying medium. B.avium and B. bronchiseptica have colonies of similar appearance on
MacConkey agar.
 132

Smith- Baskerville (SB) medium contains the pH indicator bromothymol blue and the agar is
green at pH 6.8. The colonies of B. avium and B. bronchiseptica, after 24 hours incubation, are small
(0.5 mm in diameter or less), blue colonies with a lighter blue (alkaline) reaction in the medium around
them. After 48 hours' incubation, the colonies are 1.0-2.0 mm in diameter, blue or blue with a green
centre and the surrounding medium is blue.

BIOCHEMICAL REACTIONS

B. bronchiseptica is positive to the oxidase, catalase, citrate, urease and nitrate tests. It is
motile and carbohydrates are not utilized. B. avium and Alcaligenes faecalis have similar reactions to
those of B. bronchiseptica but are urease-negative and nitrate-negative.

PATHOGENESIS AND PATHOGENICITY

The bordetellae exhibit phase changes, which correlate with virulence and are identifiable by
colonial appearance. Virulence is mediated by several factors including a filamentous haemagglutinin,
pertactin and fimbriae which allow attachment to the cilia of the upper respiratory tract. These factors
are only expressed in the virulent phase (phase 1) and are controlled by a virulence gene regulatory
system. After repeated subculture, isolates change to an avirulent form (phase 4) and colonies exhibit
a different morphology which reflects alterations in bacterial structure. Phases 2 and 3 are poorly
defined.

The tracheal cytotoxin inhibits ciliary motility and tracheobronchial clearance. In addition,
B.bronchiseptica produces an adenylate cyclase-haemolysin which primarily targets phagocytic cells.
This toxin is unique as it has the features of a repeats-in-structural-toxin but with an extra domain for
an adenylate cyclase enzyme. Although B.avium lacks the filamentous haemagglutinin, the organism
does produce a haemagglutinin, which specifically agglutinates guinea-pig red cells and correlates
with pathogenicity for turkey poults. Two other toxins, dermonecrotic toxin and osteotoxin, may be of
significance in atrophic rhinitis by contributing to nasal turbinate atrophy.

DIAGNOSTIC PROCEDURES

Specimens for laboratory examination include nasal swabs, tracheal aspirates and exudates.
Bordetellae are cultured on blood agar and MacConkey agar or on selective media. Plates are
incubated aerobically at 37°C for 24 to 48 hours.

Identification criteria for isolates:

- Colonial appearance on blood agar or selective media.

- Growth on MacConkey agar.

- Biochemical profile.

- Slide baemagglutination tests correlating with the virulence of isolates.

- Serological tests which have been developed are of limited diagnostic value.

CLINICAL INFECTIONS

Clinical signs associated with bordetellae usually relate to upper respiratory tract infection.
Young animals are most susceptible and infections in adults are usually mild or subclinical.
Predisposing factors such as stress or concurrent infections contribute to field outbreaks of disease.
Although morbidity rates may be high, mortality rates are usually low. Bordetella parapertussis, a
recognised human pathogen, has been isolated from lambs with chronic nonprogressive pneumonia.
 133

Bordetella bronchiseptica is implicated in a mild form of atrophic rhinitis in pigs and in canine
infectious tracheobronchitis (kennel cough). Oropharyngeal swabs from healthy cats may yield
B.bronchiseptica and severe bronchopneumonia associated with the organism has been reported in
kittens. Bordetella bronchiseptica may occasionally cause outbreaks of respiratory disease in rabbits
and in laboratory rodents. Bordetella avium causes turkey coryza and respiratory disease in quails.

CANINE INFECTIOUS TRACHEOBRONCHITIS

Canine infectious tracheobronchitis, also known as kennel cough, is one of the most
prevalent respiratory complexes of dogs. Although Bordetella bronchiseptica, canine
parainfluenzavirus 2 (PI-2) and canine adenovirus 2 (CAV 2) are considered to be the most important
participating pathogens, other microbial pathogens may also be involved.

Clinical signs

Clinical signs of infection with B.bronchiseptica develop within 3 to 4 days of exposure and,
without complications, persist for up to 14 days. They include coughing, gagging or retching and mild
serous oculonasal discharge. Affected dogs usually remain active, alert and non-febrile. The disease
is self-limiting unless complicated by bronchopneumonia which may develop in unvaccinated pups or
in older immunosuppressed animals.

Diagnosis

Diagnosis is based on a history of recent exposure to carrier dogs and characteristic clinical
signs. The appropriate specimen for laboratory examination is transtracheal aspiration fluid. Virulent
isolates of B.bronchiseptica haemagglutinate ovine and bovine red cells. Serology, in association with
vaccination history, may be of value for determining the involvement of respiratory viruses.

Treatment

Dogs with mild clinical signs do not require specific therapy. If coughing persists for more
than 2 weeks or if bronchopneumonia is present, antibiotic therapy may be required. Amoxicillin has
proved effective in field trials. Tetracyclines and fluoroquinolones may also be effective.

Control

Affected dogs should be isolated immediately. If predisposing factors are identified, they
should be corrected. lntranasal vaccines containing B. bronchiseptica and

PI-2 antigens induce local protective immunity and are not affected by maternal antibodies.
Modified live B. bronchiseptica vaccines decrease the severity of clinical signs but may not prevent
infection. Modified live vaccines are available for many of the viruses associated with respiratory
disease in dogs.

TURKEY CORYZA

Turkey coryza, caused by B.avium, is a highly contagious upper respiratory tract disease of
poults with high morbidity and low mortality. Infection is spread through direct contact, by aerosols
and from environmental sources. Mucus accumulates in the nares with swelling in the submaxillary
sinuses. Beak-breathing, excessive lacrimation and sneezing may be evident. Infection with B. avium
predisposes to secondary infections with bacteria such as Escherichia coli. Once E. coli becomes
established, a more serious disease with high mortality can develop.

Diagnosis

Clinical signs and gross pathological features may be indicative of the disease. Isolation and
 134

identification of B. avium from sinus and tracheal exudates is confirmatory.

Virulent isolates agglutinate guinea-pig red blood cells. Microagglutination and ELlSA
techniques may be of diagnostic value.

Treatment and control

Broad spectrum antibiotics early in the course of disease may be beneficial. Commercially
available bacterins and modified live vaccines may be used in susceptible flocks. Thorough cleaning
and disinfection of turkey houses after an outbreak of disease are essential for the elimination of B.
avium.

DISEASES AND HOSTS OF THE BORDETELLAE

Species Host(s) Disease

Bordetella pertussis Humans Classical form of whooping cough

Chimpanzees Rare case of whooping cough·like disease in captive

Animals

B. parapertussis Humans Mild form of whooping cough

Lambs Isolated from healthy and pneumonic lambs.

Significance not known
Atrophic rhinitis (with or without AR+ strains of
B. bronchiseptica Pigs Pasteurella multocida) Bronchopneumonia seen in young
pigs

Canine infectious tracheobronchitis (kennel cough) with or

Dogs without concurrent respiratory viruses.
Secondary invader in canine distemper

'Snuffles' like syndrome with upper respiratory tract

infection, bronchopneumonia or septicaemia
Rabbits
Similar to the disease in rabbits

Guinea pigs and

rats Respiratory infections (not common)

Horses, cats Occasionally isolated from wounds and body flu ids
(presumed to be zoonotic infections)
Humans
B. avium Turkeys and Turkey coryza: rhinotracheitis and sinusitis in young
less commonly poults. Morbidity high but mortality low
other birds
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MORAXELLA BOVIS

Moraxella bovis occurs as short (1.0 to 1.5 x 1.5 to 2.5 µm), plump Gram-negative rods or,
occasionally, cocci which typically occur in pairs. This organism is non-motile, aerobic and usually
catalase-positive and oxidase-positive. Although proteolytic, it is unable to utilize sugars. Growth,
which is enhanced by the addition of blood or serum to media, does not occur on MacConkey agar.
The optimal temperature for growth is 33-35°C. Most M.phenylpyruvica strains will grow on
MacConkey agar. But M.bovis and M.lacunata are unable to do so.

Virulent strains, when isolated from cases of infectious bovine keratoconjunctivitis, are
fimbriate, haemolytic and grow into the agar. Species, other than M.bovis, which are periodically
isolated from clinical specimens, are generally regarded as non-pathogenic.

USUAL HABITAT

Moraxella bovis is found on mucous membranes of carrier cattle. The organism is

susceptible to desiccation and is short-lived in the environment. It can survive for up to 72 hours in
the salivary organs and on the body surface of flies, which can act as vectors.

COLONIAL APPEARANCE

On blood agar after 48 hours' incubation the colonies of M.bovis are flat, round, small (1 mm
diameter), greyish-white and friable, surrounded by a narrow zone of complete haemolysis. The
appearance is not unlike that of a beta-haemolytic streptococcus. New isolates are often piliated and
erode the agar, sinking into it. Colonial growths will autoagglutinate when suspended in saline. On
subculture, colonial variation is common, pili are no longer formed and the colonies are butyrous and
less likely to autoagglutinate. Some colonies can become non-haemolytic. The strains of M. hovis
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(M.equi) isolated from horses are non-haemolytic even on primary isolation. M.lacunata and M.
phenylpyruvica are non-haemolytic on blood agar and some strains of M. phenylpyruvica will grow on
MacConkey agar.

BIOCHEMICAL REACTIONS

None of the moraxellae attack carbohydrates with the formation of acid. They are non-motile,
indole-negative and all are sensitive to penicillin. M.bovis will slowly pit a Loeffler serum slope, is
relatively salt tolerant and grows on media containing 5 per cent Nacl. Litmus milk medium inoculated
with M.bovis becomes alkaline (blue) and develops three zones: a deep blue upper layer, a soft blue
curd in the centre with the bottom white (reduced) and coagulated. The Moraxella species have also
been differentiated by the analysis of the cellular fatty acid.

CLINICAL INFECTIONS

Moraxella bovis causes infectious bovine keratoconjunctivitis, an important ocular disease of

cattle which occurs worldwide. Variants of M.bovis have been isolated from horses with conjunctivitis.

INFECTIOUS BOVINE KERATOCONJUNCTIVITIS

Infectious bovine keratoconjunctivitis (IBK), sometimes referred to as 'pink-eye' or New Forest

disease, is a highly contagious condition affecting the superficial structures of the eyes, usually in
animals under 2 years of age. The disease causes economic losses arising from decreased weight
gain in beef breeds, loss of milk production, short term disruption of breeding programmes and
treatment costs.

There appears to be an age-related immunity, probably as a result of previous exposure.

Asymptomatic carrier animals harbour M.bovis in the nasolacrimal ducts, nasopharynx and vagina.
Transmission can occur by direct contact, by aerosols and through flies acting as vectors.

PATHOGENESIS AND PATHOGENICITY

The virulence of M.bovis is attributed to fimbriae, which allow adherence of the organisms to
the cornea, circumventing the protective effects of lacrimal secretions and blinking. Two types of
fimbriae are recognized, namely, Q fimbriae (pili) which are specific for colonization and ‘I’ fimbriae
which allow local persistence of infection. Fimbrial antigens stimulate type-specific protective
immunity.

During bacterial replication, haemolysin and other lytic enzymes such as fibrolysin,
phosphatase, hyaluronidase and aminopeptidase are produced. Lipopolysaccharides, associated with
O antigens, also appear to play a role in virulence. The haemolysin is a calcium-dependent, pore-
forming cytolysin which damages the cell membranes of neutrophils. Release of hydrolytic enzymes
from neutrophils on the corneal surface contributes to breakdown of its collagen matrix.

Strains which lack either haemolysin or fimbriae are avirulent. Isolates from carrier animals
are often non-haemolytic and non-fimbriate but reversion to virulence can occur. It has been
suggested that the deficiency of lysozyme in the lacrimal secretions of cattle may account for their
susceptibility to M.bovis.

CLINICAL SIGNS

Infectious bovine keratoconjunctivitis initially manifests as blepharospasm, conjunctivitis and

lacrimation. Progression of the condition through keratitis to corneal ulceration, opacity and
abscessation may occasionally lead to panophthalmitis and permanent blindness. Following
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ulceration, vascularisation extends from the limbus and stromal oedema develops. There may be
weakening of the cornea with the development of coning. In most mild cases, the cornea heals within
a few weeks although there may be permanent scarring of the structure. Some carrier animals may
exhibit persistent lacrimation. Following infection with a virulent strain of M.bovis, neutralizing
antibodies develop which are active against haemolysin produced by other strains. In contrast,
antibodies which block fimbrial-mediated adherence are type-specific and exposure to M. bovis
possessing a different fimbrial type may result in disease.

DIAGNOSTIC PROCEDURES

 The disease characteristically affects a number of animals in a herd. Lacrimal secretion is the
most suitable specimen for laboratory examination. Because M.bovis is extremely
susceptible to desiccation, specimens must be processed promptly. For transportation,
swabs of lacrimal secretions should be placed in 1 to 2 ml of sterile water. Ideally, specimens
should be cultured within 2 hours of collection.

 A fluorescent antibody technique for demonstrating M.bovis in smears from lacrimal

secretions is available.

 Specimens should be cultured on blood agar and MacConkey agar and incubated aerobically
at 37°C for 48 to 72 hours.

 Identification criteria for isolates:

- Round, small, shiny, friable, colonies appear after 48 hours. Colonies of virulent
strains are surrounded by a zone of complete haemolysis and are embedded in the
agar.

- No growth occurs on MacConkey agar.

- Cultures of virulent strains autoagglutinate in saline.

- Smears from colonies reveal short Gram-negative rods in pairs.

- Reactions in the catalase and oxidase tests are positive.

- A Loeffler's serum slope may be pitted after 10 days.

Isolation

Lacrimal secretions should be inoculated as soon as possible after collection on blood agar and
incubated at 35 °C for 48-72 hours. The inoculation of a MacConkey plate is useful to gauge the
degree of contamination by other Gram- negative bacteria. M. bovis is unable to grow on MacConkey
agar.

TREATMENT

Antimicrobial therapy should be administered subconjunctivally or topically early in the

disease.

CONTROL

Fimbriae-derived bacterins which are available commercially in some countries are of

uncertain efficacy. Management-related methods are important in the control of IBK. These include
isolation of affected animals, reduction of exposure to mechanical irritants, the use of insecticidal ear
tags and the control of concurrent diseases, such as infectious bovine rhinotracheitis or Thelazia
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infestation. The prophylactic use of intramuscular oxytetracycline can be considered for animals at
risk. Animals which are blind should be housed. Vitamin A supplementation may be beneficial.

Summary of the diseases and sites of isolation of the Moraxella species

Species Host Disease and/or site of isolation

Moraxella bovis Cattle Infectious bovine keratoconjunctivitis ['pink eye'

or 'New Forest
disease· (UK)]

Horses Rare isolates from horses with conjunctivitis

M.lacunata Many animal species Isolated from guinea·pigs, aborted equine

foetuses, goats with viral pneumonia and
encephalitis, a goat with septicaemia and from
various pathological specimens from dogs and
pigs. Its role in disease processes in animals is
not known

Humans May cause conjunctivitis

M. phenylpyruvica Sheep and cattle Recovered from the urinogenital tract and brain

Isolated from the urinogenital tract

Pigs
Obtained from the intestinal tract
Goats
Pathogenicity for animals is unknown
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BACTEROIDES

Bacteroides spp are non-spore forming gram-negative bacilli that are part of the human
resident flora. Microbiologically, they are distinguished from other genera by growth in 20% bile. At
present, the Bacteroides fragilis group consists of ten species: B. fragilis (the most frequent
isolate), B. distasonis, B. thetaiotaomicron, B. vulgatus, B. ovatus, B. eggerrthii, B. merdae, B.
stercoris, B. uniformis, and B. caccae. Since 1990, many organisms previously designated
as Bacteroides have been reclassified (see chapter on Anaerobes other
than Bacteroides). Bacteroides, the predominant genus in the human intestine, are important in
numerous metabolic activities and may provide some level of protection from invasive pathogens. All
10 species are usually isolated from the colon, although infections caused by or associated with them
can include virtually any organ.

CHARACTERISTICS

The gram-negative Bacteroides spp. or closely related genera are capsulated obligatory
anaerobic bacilli that are non-spore forming, pale-staining, and some are motile by peritrichous
flagella, while other taxa are non-motile. Bacteroides, Parabacteroides, Odoribacter are generally bile
resistant, distinguished from genera which are bile sensitive. They are normally commensal, found in
the intestinal tract of humans (mouth, colon, urogenital tract) and other animals. Many cultures of
Bacteroides strains display brown to black pigmentation on blood agar media caused by esculin
hydrolysis.

Morphology

In general, all strains were stained poorly with safranin, but well with dilute carbolfuchsin.
Motility was not observed in any strains.

The morphology of Bacteroides fragilis in fresh cultures and in pus is not especially
characteristic. In this form, the bacillus measures 0.5 by 2.0 to 2.5 micra. Older cultures tend to
develop larger forms (0.6 to 0.8 by 2.0 to 9.0 micra) but even in this state the organism cannot be
distinguished from aerobic Gram-negative bacilli, by its morphology.

Bacteroides funduliformis, on the other hand, is characteristic morphologically because of its

extreme pleomorphism. In the necrotic centers of lesions, growth occurs in such dense clumps of
small bacillary forms that it is difficult to pick out the individual bacilli. Bipolar staining is a prominent
feature and gives the bacilli a diplococcus-like appearance. Filaments 1.0 by 10.0 micra have been
observed in microscopic sections of the granulating periphery of abscesses. On solid mediums, long
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unbranched filaments develop after six or seven days of cultivation. In solid mediums, there are
fusiform swellings 4.0 to 5.0 micra wide.

In brain broth the earliest forms are coccobacillary, similar to those seen in pus, or rarely,
short curved "comma" shaped rods 0.3 to 0.5 by 2.0 to 4.0 micra. Within two days the characteristic
"funduliform," "ball," "leukocyte," or "thetoid" forms appear, together with straight bipolar staining rods
which vary in size from coccobacillary up to filaments 20.0 to 40.0 micra long. The ball-shaped
organisms (1.5 to 3.0 micra in diameter) seem to be formed by bacilli, swollen so that the bipolar dark
staining beads appear as crescentic caps opposite each other on the ball.

Bacteroides A is slightly shorter and more plump (1.0 by 2.0 micra) than Bacteroides fragilis.
Bipolar staining in day-old broth cultures is prominent. A characteristic feature of this bacillus is its
tendency to develop tremendously swollen forms even in forty-eight hour cultures. These swollen
bacilli are less definite in outline than the "funduliforms" of Bacteroidesfunduliformis, and look like
bacteria which are undergoing lysis.

Grampositive granules are present in the bacilli only in very fresh cultures. The strain of
Fusiformis dentium was Gram-positive only in the pus from which it was obtained. The long filaments
of the Actinomycete develop in cultures seven or eight days old. Fresher cultures contain only
bacillary forms. The anaerobic diphtheroid loses its diphtheroid appearance when it becomes Gram-
negative after three to four days of incubation. Its morphology is not characteristic.

BIOLOGIC CHARACTERISTICS

In general, all strains have been able to survive in glucose brain broth for several weeks at
room temperature. None of the organisms survive long, however, if exposed to air. They are able to
grow in an atmosphere of nitrogen. Although all strains seem to grow better in brain broth which
contains fermentable carbohydrate, growth is not improved on solid mediums by the addition of
higher concentrations of glucose.

Bacteroides fragilis grows on the surface of plain blood agar as very fine, transparent, moist,
grayish white, fimbriated or spreading colonies, without production of hemolysis. Isolated surface
colonies do not exceed 2 mm. in diameter. On this medium, growth is seldom perceptible before five
days of incubation.

Growth on hormone blood agar is more profuse and more rapid, and the colonies are yellow
and opaque. Deep colonies are yellow, opaque, and lens-shaped. They do not exceed 1 mm.

Growth of Bacteroides fragilis in glucose-brain broth takes place in twenty-four hours and
produces a diffuse clouding of the medium. A characteristic feature of the growth in fermentable
carbohydrate mediums is the slight but definite production of gas.

In an anaerobic jar, growth is obtained in peptone-water sugar mediums, but not in plain broth.
The biologic reactions of the first three strains are uniformly similar, and those of the fourth strain,
which is a subspecies, vary only slightly.

The production of indol by Bacteroides fragilis is listed as either negative or positive. For the
most part, tests for indol were negative but we have encountered an occasional faintly positive
reaction in fourteen-day old cultures.

Bacteroides funduliformis grows on plain blood agar with as much difficulty as does
Bacteroides fragilis, but the colonies are larger (3 mm.) and more opaque. The centers of the colonies
are grayish-white and opaque, the borders are fimbriated and may spread to coalesce with other
colonies on a sufficiently moist medium. On slants, when the moisture has drained from the surface,
colonies are discoid, discrete, and have slightly raised centers and edges.
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The colonies when they first appear are fairly moist, but after incubation for ten days they are
hard and dry, and can be picked entire from the medium. Deep colonies are lens-shaped or triangular
and measure 2 mm. in their greatest diameters. They occasionally produce gas in blood agar which
contains glucose, particularly if the medium is not too stiff. The hemolytic action of this species is an
important characteristic. The radius of the zone of hemolysis measures 4 to 5 mm.

In brain broth, Bacteroides funduliformis grows in clumps at the bottom of the tube. In fresh
cultures, a few bacilli are diffused throughout the broth, but they are scarcely sufficient in number to
produce appreciable clouding. Often, the only indication of growth is the presence of bubbles of gas
caught in the particles of brain at the bottom of the tube.

GENERAL CHARACTERISTICS

 Gram negative anaerobic rod

 Shape: Pleuromorphic

 Size: (0.5-1.5)µm wide and (2-6)µm long

 Non motile except B. polypragmatus, B. xylanolyticus

 Non capsulated except fragilis

 Non spore forming

CLASSIFICATION

On the basis of medical importance bacteriodes is classified into two group;

1. Bacteroides fragilis group:

Examples:

 B.fragilis

 B. distasonis

 B. ovatus

 B. thetaiotaomicron

 B. vulgatus

 B. idgatus

 B. uniformis

 B. tiariabilisleggerthii

 B. splanchnicus

These are commensals of GI tract

2. Bacteroides melaninogenicus group

Examples:
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 B. melaninogenicus sub spp intermedius

 B. ruminicola

 Now the name has changed to Prevotella melaningenica

 These are Norma flora of URT, GIT, vagina

SUSCEPTIBILITY TO DISINFECTANTS

More specific information on Bacteroides spp. is not available, but most bacteria have been
shown to be susceptible to low concentration of chlorine, 1% sodium hypochlorite, 70% ethanol,
phenolics such as orthophenylphenol and ortho-benzyl-paua-chlorophenol, 2% aqueous
glutaraldehyde, iodine, formaldehyde, and peracetic acid (0.001% to 0.2%).

PHYSICAL INACTIVATION

Information specific to Bacteroides and like genera is not available, but most bacteria can be
inactivated by moist heat (121°C for 15 min - 30 min) and dry heat (160-170°C for 1-2 hours).

SURVIVAL OUTSIDE HOST

Bacteroides and like genera have been detected in faeces infected water by PCR for at least 2
weeks at 4°C; 4 to 5 days at 14°C; 1 to 2 days at 24°C; and 1 day at 30°C.

EPIDEMIOLOGY

Bacteroides fragilis are endogenous organisms of the GI tract. Spread of strains among
patients is not known, although this topic has not been well studied. Thus, infections due to this
organism are most likely caused by endogenous strains.

MODE OF TRANSMISSION

Infection results from displacement of Bacteroides spp. or closely related genera from
normal mucosal location as a result of trauma such as animal/human bites, burns, cuts, or
penetration of foreign objects, including those involved in surgery. There is no evidence that
organisms are invasive on their own.

PATHOGENESIS

Bacteroides fragilis is the most common opportunistic pathogen of Bacteroides spp. Spread
to bloodstream (bacteremia) is more common for B. fragilis than any other anaerobe. Deep pain and
tenderness below the diaphragm are typical of B. fragilis infection. Widespread intra-abdominal
abscesses may be associated with fever and abdominal pain.

Multiple virulence factors have been implicated in the pathogenesis of this organism. They
include the capsular polysaccharide (which inhibits opsonophagocytosis and promotes abscess
formation), pili and fimbriae (promotes adherence), and production of a number of different enzymes
(hyaluronidase, hemolysin, peroxidase, collagenase, protease, heparinase, and neuraminidase). In
addition, superoxide dismutase and catalase also considered virulence factors. These enzymes
defend B. fragilis against oxygen radicals and increase aerotolerance.

Bacteroides spp. represents an important anaerobic bacterial genus associated with human
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infections. In combination with other facultative/strict anaerobes, they are responsible for the
majority of localized abscesses within the cranium, thorax, peritoneum, liver, and female genital tract.
They can cause pulmonary abscesses when naturally-occurring oropharangeal Bacteroides and
closely related genera are aspirated into the lung. These taxa can lead to many types of diseases,
some of which can be fatal, including noma (cancrum oris), human apical periodontitis, endocarditis,
pelvic inflammatory disease, suppurative thrombophelebitis, and wound infections. Organisms from
oral flora also have a role in dental abscesses and infectivity of human bites.

CLINICAL MANIFESTATIONS

The hallmarks of nearly any infection involving Bacteroides spp include abscess formation,
and they are frequent isolates in polymicrobial infections. Typical sites of polymicrobial infections
involving Bacteroides include the abdomen and pelvis, perirectal, skin and soft tissue, and solid
organs. Although isolation of Bacteroides spp as the sole pathogen can occur, it is unusual. Infections
where single organism isolation is most commonly associated with Bacteroides include endocarditis,
meningitis, septic arthritis and osteomyelitis.

LABORATORY DIAGNOSIS

Bacteroides fragilis may be isolated as a single agent, such as in blood cultures, or more
typically from mixed infections. The organism is aerotolerant, but requires an anaerobic environment
to propagate. Simple identification from blood cultures includes Gram stain and growth on blood agar
and Bacteroides-bile-esculin (BBE) agar for isolation and presumptive identification ofBacteroides
fragilis group (as well as Bilophila wadsworthia). B. fragilis will appear as dark colonies with brown-
black halos on BBE agar due to the hydrolysis of esculin. B. fragilis can be further presumptively
identified by resistance to kanamycin, vancomycin and colistin, using a disk test, and will grow in 20%
bile, produce catalase (most strains), and is variably indole positive.

Laboratory diagnosis:

Samples: pus, exudates, biopsy

1. Microscopy:

Gram negative, non motile, non sporing pleuromorphic rod

2. Culture:

i) Blood Agar (BA):

Kanamycin or neomycin Blood agar selective for Anaerobes

Inoculates anaerobically at 37C for 48 hours

Fragilis Form non haemolytic grey colony of 1-3 mm diameter

Melanogenicus form black brown haemolytic colony in 3-5 days

ii) Bactrroides Bile Esculin Agar (BBE):

Hydrolyses esculin,

Colony surrounded by dark zone

3. Biochemical tests for fragilis

i) Esculin hydrolyses: positive (+)

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ii) Indole: negative (-)

iii) Urease: Negative (-)

iv) Catalase: Positive (+)

v) Oxidase : variable (+/-)

vi) Fermentative:

 Glucose +ve

 Lactose + ve

 Maltose + ve

 Sucrose + ve

 Rhamnose - ve

 Arabinose - ve

 Salicin - ve

 Trehalose - ve

vii) Bile resistant: grow in 20% bile

viii) Thioglycolate with bile: Positive (+)

ix) Beta lactamase production: fragilis is resistant to penicillin

4. Serology

5. PCR

DRUG RESISTANCE

Resistance against antibiotics is increasingly common, and frequently observed with penicillin,
ampicillin, cephalothin, tetracycline, piperacillin, chloramphenicol, kanamycin, colistin, rifampicin,
vancomycin, and the aminoglycosides. The B. fragilis group is commonly resistant to expanded and
broad spectrum cephalosporins, including β-lactamase-resistant drugs such as cefoxitin, and
clindamycin.

Strain resistance to imipenem and metronizadole, although it has been detected worldwide,
are rarely encountered. Resistance to quinolones is increasing. Some members of B. fragilis group
have shown resistance to ampicillin-sulbactam and amoxicillin-clavulanante combination therapies.
Isolates often produce β-lactamase, often making penicillin based antibiotics ineffective.
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DICHELOBACTER NODOSUS

Many non-spore-forming, anaerobic, Gram-negative bacteria cause opportunistic mixed

infections, often in association with facultative anaerobes. Synergistic interactions between the
organisms in these mixed infections are common. Fusobacterium species and bacteria formerly
referred to as Bacteroides species account for more than 50% of the anaerobic organisms isolated
from these infections.

USUAL HABITAT

Nan-spore-forming, Gram-negative anaerobes are often found on muwus membranes,

particularly in the digestive tract, of animals and man. They are excreted in the faeces and they can
survive for short periods in the environment. Dichelobacter nodosus, a primary pathogen of the
epidermal tissues of the hoof region of ruminants, survives for less than 4 days in mud.

DIAGNOSTIC PROCEDURES

In order to ensure that isolates of anaerobes are aetiologically significant, specimens for
isolation procedures should be obtained by direct sampling from discharges or lesions and by supra
pubic puncture in urinary infections.

Specimens should be processed promptly after collection. Commercial kits and transport
media are available for specimens from suspected anaerobic infections. In the core of a tissue
specimen over 2-3 cm, an anaerobic microenvironment is usually maintained. Samples of fluid in a
syringe remain suitable for anaerobic culture if air is expelled from the syringe and the needle is
plugged.

Anaerobic jars with an atmosphere of hydrogen and 10% CO2 are used for incubating cultures
at 37°C for up to 7 days.

Enriched blood agars for the isolation of anaerobes are supplemented with 5 to 10% ruminant
red cells, yeast extract, vitamin K and haemin. Selective media can be prepared by adding appropriate
antimicrobial agents. Media must be pre-reduced by storing them in an anaerobic atmosphere for at
least 6 hours before inoculation. Liquid media, such as cooked meat broth or thioglycollate medium
supplemented with vitamin K and haemin, are useful for subculturing but are unsuitable for primary
isolation.

Special selective media are required for the isolation of Dichelobacler nodosus from ruminant
footrot. In some media formulations, powdered ovine hoof is added to promote enhanced growth.

PATHOGENESIS AND PATHOGENICITY

Non-spore-forming anaerobes usually exert pathogenic effects when anatomical barriers are
breached allowing invasion of underlying tissues. They replicate only at low or negative reduction
potentials (Eh). Most of those involved in opportunistic infections produce superoxide dismutase
which allows them to survive in oxygenated tissues until the Eh reaches levels favouring their growth.

Tissue trauma and necrosis followed by multiplication of facultatively anaerobic bacteria can
lower Eh levels to a range suitable for the proliferation of non-spore-forming anaerobes. Most
infections involving these organisms are mixed. Two or more bacterial species, interacting
synergistically, may produce lesions which the individual organisms cannot. A relevant example of
this type of synergism is the production by Arcanobacterium pyogenes of a heatlabile factor which
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stimulates F. Necrophorum replication. In turn, F. necrophorwn produces a leukotoxin which

correlates with the strain virulence and aids survival of A. pyogenes.

Synergism between F. necrophorum and Dichelobacter nodosus is important in the

pathogenesis of ruminant pedal lesions. In this instance, F. Necrophorum facilitates tissue invasion
by D. nodosus and is itself stimulated by a growth factor elaborated by D.nodosus.

Three biotypes of F. necrophorum are recognised. Biotype A, designated F. necrophorum

subspecies necrophorum has greater haemolytic activity and is more virulent than biotype B, F.
necrophorum subspecies funduliforme. Biotype C, reclassified as F. Pseudonecrophorum appears to
be non-pathogenic. Characteristics of D. nodosus which correlate with its ability to damage tissues
include the production of thermostable proteases and elastase and the presence of agarolytic activity
on agar-based media containing powdered hoof.

CLINICAL INFECTIONS

Calf diphtheria

This condition usually presents as necrotic pharyngitis or laryngitis in calves less than 3
months of age. The aetiological agent, F. necrophorum, can enter through abrasions in the mucosa of
the pharynx or larynx often caused by ingestion of coarse feed. Clinical signs include fever,
depression, anorexia, excessive salivation, respiratory distress and a foul smell from the mouth.
Untreated calves may develop a fatal necrotizing pneumonia. Treatment with potentiated
sulphonamides or tetracyclines early in the course of the disease is usually effective.

Bovine liver abscess

Hepatic abscessation in cattle, secondary to rumenitis, is encountered most commonly in

feedlot animals. The feeding of rations high in carbohydrates and the resulting rapid intraruminal
fermentation can lead to the development of ulcers. Fusobacterium necrophorum together with other
anaerobes and Arcanobacterium pyogenes invade the tissues, and occasional emboli which reach the
liver via the portal vein initiate abscess formation.

Affected cattle rarely show clinical signs and lesions are usually detected at slaughter.
Management techniques in feedlots should be aimed at reducing the incidence of rumenitis.
Chlortetracycline in feed during the finishing period can reduce the prevalence of liver abscess.

Necrotic rhinitis of pigs

This sporadic condition, primarily affecting young pigs, is characterised by suppuration and
necrosis of the snout as a result of infection with F. necrophorum, often in association with other
anaerobes.

These organisms enter through abrasions in the nasal mucosa. Signs include swelling of the
face, sneezing and a foul-smelling nasal discharge. In chronic infections, involvement of the nasal and
facial bones can result in permanent facial deformity ('bull nose'). Potentiated sulphonamides
administered early in the course of the infection may be beneficial.

Thrush of the hoof

This necrotic condition of the horse's hoof is associated with poor hygiene, wet conditions
and lack of regular cleaning of the hooves. Infection with F. necrophorum, secondary to hoof damage,
results in a localized inflammatory response. Thrush, which commonly affects the hind feet, is
characterized by a foul-smelling discharge in the sulci close to the frog. The aim of therapy is to
encourage regeneration of the frog by providing dry, clean stabling, regular attention to the hooves
 147

and exercise.

Black spot of bovine teats

Black spot or black pox of the teat orifice and sphincter of dairy cows presents as a localized
area of necrosis with black scab formation due to invasion by Fusobacterium necrophorum. The
condition can contribute to stenosis of the sphincter and may predispose to mastitis.

SPIROCHAETES

The order Spirochaetales contains two families, Leptospiraceae and Spirochaetaceae. It

comprises spiral or helical bacteria (spirochaetes) which share some unique morphological and
functional features. Members of the order are motile by means of endoflagella which are located
within the periplasm.

The spirochactes are slender, motile, flexuous, unicellular, helically coiled bacteria ranging
rrom 0.1 -3.0 µm in width. The outer sheath, the outermost layer of a spirochaete cell, is a multi
layered membrane that completely surrounds the peri plasmic flagella (axial filament) and the helical
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protoplasmic cylinder. The cylinder consists or the nuclear material, cytoplasm, cytoplasmic
membrane and the peptidoglycan portion of the cell wall.

The periplasmic flagella are wrapped around the cylinder and are in the peri plasmic space of
these Gram-negative bacteria. One end of each flagellum is inserted near a pole of the protoplasmic
cylinder and attached by plate-like structures called insertion discs. The distal end of each flagellum is
not inserted and extends to the centre of the cell and may overlap the flagellum from the opposite end.
The periplasmic flagella facilitate the motility of the bacteria in viscid environments.

Leptospira species

Members of this species (leptospires) are motile helical bacteria (0.1 x 6 to 12 µm) with hook-
shaped ends. Although cytochemically Gram-negative, they do not stain well with conventional
bacteriological dyes and are usually visualized using dark-field microscopy. Silver impregnation and
immunological staining techniques are used to demonstrate leptospires in tissues. Leptospirosis,
which can affect all domestic animals and humans, ranges in severity from mild infections of the
urinary or genital systems to serious systemic disease.

Culture

The media used for the culture of leptospires include Korthof and Stuart broths, Fletcher
semisolid medium, EMJH and Tween 80-albumin medium (OAC) that are liquid but can be made semi
-solid by the addition of 1.5g agar/litre of medium. Contaminated samples (or cultures) can be filtered
through a 0.45 µm bacteriological filter and inoculated into a medium contain ing 5-fluorouracil at 200
µg/ml. The media can be inoculated with 1-3 drops of carefully taken urine within a few minutes of
collection, or urine dilUled 1: 10 with 1 per cent BSA, as soon as possible after collection.

A 10 per cem tissue suspension in 1 per cent BSA (1-2 drops) or a few drops of oxalated or
heparinised blood can also be inoculated into media. The cultures are incubated at 30°C for up to 8
weeks. A drop of the culture is examined by darkfield microscopy once weekly. Leptospira hardjo is
one of the slowest growing serovars and bratislava is difficult to culture in laboratory media.

USUAL HABITAT

Leptospires can survive in ponds, rivers, surface waters, moist soil and mud when
environmental temperatures are warm. Pathogenic leptospires can persist in the renal tubules or in
the genital tract of carrier animals. Although indirect transmission can occur when environmental
conditions are favourable, these fragile organisms are transmitted most effectively by direct contact.

PATHOGENESIS AND PATHOGENICITY

Leptospires gain entry through mucous membranes or damaged skin from direct or indirect
contact. After epithelial penetration there is haematogenous spread with localisat ion and
proliferation in parenchymatous organs, particularly the liver, kidneys, spleen and sometimes
meninges. In the kidneys the organisms reach and localise in the lumen of proximal convoluted
tubules. Penetration and multiplication in the foetus can occur in pregnant animals leading to foetal
death and resorption, abortion or weak offspring. The foetus, if infection occurs in the third trimester,
can produce specific antibodies and may overcome the infection.

Antibody production in infected animals begins a few days after the onset of leptospiraemia.
The leptospires tend to persist in sites such as renal tubules, eyes and uterus where antibody activity
is minimal.

Leptospires damage vascular endothelium resulting in haemorrhages. Serovars in the

serogroups Autumnalis, Grippotyphosa, Icterohaemorrhagiae and Pomona produce a haemolysin that
 149

is probably responsible for the haemoglobinuria (redwater) in young calves infected with these
serovars. Cytotoxic protein is produced by virulent strains but the role of the toxin is unknown.
Virulence varies between the serovars and between two genotypes of L. interrogans serovar hardjo
known as hardjobovis and hardjoprojitno.

DIAGNOSTIC PROCEDURES

Diagnosis of leptospirosis in maintenance hosts usually requires screening of a defined

population. Clinical signs, together with a history suggestive of exposure to contaminated urine, may
suggest acute leptospirosis.

Organisms may be detected in fresh urine by dark-field microscopy, but this technique is
relatively insensitive. Slow-growing serovars such as hardjo may require incubation for six months in
liquid media at 30°C. Commonly, EMJH (Ellinghausen, McCullough, Johnson and Harris) medium
based on 1% bovine serum albumin and Tween 80 is used for isolation. Isolates should be identified
using DNA profiles and serology.

Fluorescent antibody procedures are often used for the demonstration of leptospires in
tissues. Suitable tissues include kidney, liver and lung. Silver impregnation techniques can also be
used for demonstration of leptospires.

DNA hybridization, PCR, magnetic immunocapture PCR and immunomagnetic antigen capture
systems have also been developed for the demonstration of leptospiral infection in tissues and urine.
The standard serological reference test, the microscopic agglutination test, is potentially hazardous
because it involves mixing live culture growing in liquid medium with equal volumes of doubling
dilutions of test serum.

CLINICAL INFECTIONS

Leptospirosis in cattle and sheep

Cattle are maintenance hosts for L. borgpetersenii serovar hardjo and there is increasing
evidence that this serovar is also host-adapted for sheep. Leptospira interrogans serovar hardjo is
also host-adapted for cattle. Although L. interrogans serovar hardjo appears to cause only sporadic
cases of disease in cattle, it may be more virulent than L. borgpetersenii serovar hardjo. Susceptible
replacement heifers, reared separately and introduced into an infected dairy herd for the first time at
calving, may develop acute disease with pyrexia and agalactia affecting all quarters. Infection may
also result in abortions and stillbirths.

If management practices allow exposure to infection and the subsequent development of

immunity before breeding age, reproductive problems may not develop. Agalactia caused by
leptospiral infection can be confirmed by demonstrating a rising antibody titre in paired serum
samples. Infection with serovar hardjo in sheep, particularly in intensively managed lowland flocks,
can cause abortions and agalactia. Dihydrostreptomycin or amoxycillin can be used for reducing or
eliminating urinary excretion of the organisms.

Both monovalent and multivalent inactivated vaccines, which are commercially available may
not always be effective. Serovars incorporated into vaccines should be those which are associated
with disease in a particular region. Infection with serovarspomona, grippotyphosa, and
icterohaemorrhagiae can cause serious disease, particularly in calves and lambs. Infection is usually
accompanied by pyrexia, haemoglobinuria, jaundice and anorexia. Extensive renal damage with
resultant uraemia often precedes death. Vaccination is used for control of serovar pomona which is
an important cause of bovine abortion in some countries.

LEPTOSPIROSIS IN DOMESTIC ANIMALS

 150

Host Disease syndrome

Cattle • Subclinical with or without leptospiruria

• Milk·drop syndrome, with or without any other clinical signs (often
hardjo)
• Abortion and neonatal mortality: abortion 'storms' (pomona) and
sporadic abortions (hardjo)
• Infertility (often hardjo)
• Haemoglobinuria, jaundice and fever in calves and, less commonly, in
young adults.
• Serovars commonly involved are pomona, grippotyphosa and
icterohaemorrhagiae.
• Occasionally, some animals show signs of meningitis

Pigs • Subclinical, often with leptospiruria: especially with pomona. Pigs are
considered to be the maintenance host for this serovar
• Fever and focal non-suppurative mastitis and leptospiruria
• Infertility, abortions and stillbirths: often canicola, pomona or
icterohaemorrhagiae
• Fever, anorexia, jaundice, haemoglobinuria and high mortality in young
pigs: often icterohaemorrhagiae

Dogs • Subclinical with leptospiru ria: often canicola

• Acute haemorrhagic disease: high fever, vomiting, prostration and often
early death; usually icferohaemorrhagiae
• Less acute icteric type: intense icterus, depression, fever, haemorrhages
with blood in faeces and urine; canicola or icterohaemorrhagiae
• Uraemic type: uraemia associated with extensive kidney damage,
ulcerative stomatitis and uraemic breath. Death occurs in a high
percentage of cases. These severe signs can occur 1-3 years after the
initial infection: often canicola
• Rarely a chronic, active hepatitis: seen in a grippotyphosa infection

• Recurrent iridocyclitis (‘periodic ophthalmia' or 'moon blindness') which

can result in blindness. Aetiology has not been conclusively determined
Horses • Occasionally abortion with foetuses of 6 months to term
• Rarely fever, anorexia, depression and icterus

• Mainly subclinical infections with leptospiruria: serovars such as hardjo

• Occasionally, acute leptospirosis with depression, dyspnoea,
haemoglobinuria, anaemia and high mortality in lambs: often pomona
Sheep

SERPULINA (TREPONEMA) SPECIES

Members of the genus Serpulina (Treponema) are host-associated spirochaetes found in the
oral cavity, intestinal tract and genital region of animals and humans. The cells are wider, are not as
tightly coiled as th e leptospires and can be stained by aniline dyes.

The species of veterinary significance are S. Hyodysenleriae (swine dysentery) and T.

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paraluiscunicllii (vent disease of rabbits). S. innocens, present in the faeces of pigs and dogs, is
thought to be non-pathogenic, although some workers regard S. innocens as a strain of S.
hyodysenteriae of lower virulence. S. hyodysenteriae and S. innocens are s imilar morphologically,
culturally as well as biochemically and can be grown on laboratory media. T. Paraluiscuniculi has not
been cultured in vitro.

NORMAL HABITAT

T. paraluiscliniculi produces a benign venereal disease of rabbits and is present in lesions in

the genito-perineal area of rabbits. It causes latentin fection in mice, guinea- pigs and hamsters. The
treponemes can be found in the lymph nodes of these animals. The reservoir of S. hyodysenteriae is
the intestinal tract of pigs, wild rats and mice. Recovered, asymptomatic pigs can excrete these
organisms in faeces for 3 months or more. Survival of S. hyodysenteriae in soil or voided pig faeces is
short, about 24 48 hours. Infection is by the faecal-oral route.

PATHOGENESIS

After S. hyodysenteriae enters a susceptible pig the spirochaete invades goblet cells of the
colonic mucosa, multiplies in the crypts of Lieberkuhn and causes necrosis and erosion of the
mucosal cells. The faecal material is watery and contains mucus, blood and necrotic debris. Only the
large intestine is involved and colonic malabsorption occurs. The Lipopolysaccharides of the
bacterium are thought to play a part in the pathogenicity. S. hyodysenteriae is unable to produce
dysentery in gnotobioticpigs and it appears that the pathogen requires the interaction of other
bacteria of the normal flora such as Fusobacterium necrophorum, Bacteroides vulgatus, B.fragilis and
Campylobacter coli. The disease was once thought to be due to C. coli as these curved rods can often
be seen in significant numbers in faecal smears from pigs with swine dysentery.

LABORATORY DIAGNOSIS

Specimens

Deep mucosal scrapings should be taken from a portion of the affected large intestine from a
dead pig, or rectal swabs and faeces from several live affected pigs. The numbers of S.
hyodysenteriae can sometimes be low in faeces and are difficult to see. Sections of affected colon in
10 per cent formalin should be taken for histopathology.

Direct microscopy

Various methods can be used to visualise S. hyodysenteriae:

• A portion of deep scrapings from the mucosa or faecal material is examined in a drop of water
under darkfield microscopy.

• Fixed smears of mucosal scrapings or faeces are stained by dilute carbolfuchsin (D CF) for 4-6
minutes, by Victoria blue 4-R or by a silver impregnation technique.

• Histological sections of colon can be stained by a silver impregnation stain or Victoria blue 4-R.

• Fluorescent anti body technique can be carried out on mucosal scrapings or faeces.

• Three to 5 serpulinas per high power field is considered significant. S. hyodysenteriae is 0.3-0.4 µm
in width, 6-8 µm in length, is loosely coiled with 2-4 coils and tapered ends. It is motile by flexing
movements under the darkfield. Pigs normally have other non -pathogenic treponemes such as S.
 152

innocens and smaller, tightly coiled spirochaetes in the intestinal tract that can complicate the
interpretation of darkfield examination or stained smears.

Isolation

S. hyodysenteriae is anaerobic but oxygen-tolerant and growth is enhanced by CO2. It grows

well on freshly poured, or pre-reduced, trypticase soy agar with 5 per cent blood and 400 µg/ml
spectinomycin at 42°C for 2-3 days under an atmosphere delivered by an H2 + CO2 envelope.
Increased growth has been obtained using Fastidious Anaerobe Agar (Lab M) with 5 percent sheep
blood and also by the incorporation of 1 per cent sodium RNA (BHD Chemicals) into media. Sodium
RNA can be sterilized by filtration or by autoclaving. It is a growth enhancer and also increases the
difference in haemolysis between S. hyodysenteriae and S.innocens. Colonic mucosal scrapings or
faeces can be prepared as a 1:10 suspension in saline and clarified by centrifugation at slow speed.
The supernatant can be passed, seri ally, through 0.8, 0.65 and 0.45 µm filters and this material is
used to streak the culture medium.

Identification

Colonial characteristics

After about 48 hours' incubation S. Hyodysenteriae appears as small, translucent colonies

with a zone of clear haemolysis. S. innocens is weakly beta haemolytic.

Microscopic appearance

DCF-stained smears or dark field microscopy from the colony reveals the typical helical
serpulinas.

Biochemical characteristics

S. hyodysenteriae must be distinguished from S. innocens. Belanger and Jacques (1991)

suggest that a rapid and simple differentiation can be based on the combined results of the
haemolysis, the haemolysis intensification test and an indole spot test.

• Haemotysis: a distinctive characteristic of S. Hyodysenteriae is the production of a strong

betahaemolys is that is best demonstrated on sheep blood agar. S. innocens gives a weak reaction.

• Haemolysis intensification: this test can be carried out by cutting an agar block (0.5 cm2) from a 4-
day culture and by incubating the plate for another 4 days. The intensification of haemolysis is
recognised by a lighter zone, 1- 2mm wide, along the cut line. S. hyodysemeriae gives this reaction
(ring phenomenon) but S. innocens does not.

• Indole spot test for anaerobes: the reagent is 1 percent p-dimethyl-aminocinamaldehyde in 10

percent concentrated HCl. This is used to saturate a strip of filter paper in a Petri dish and growth
from a culture of the test bacterium is smeared on the filter paper. S. hyodysemeriae gives a positive
reaction indicated by a blue colour usually within 1- 3 minutes. S. innocens is negative and the filter
paper remains pink.

Neither S. hyodysenteriae nor S. innocens are very reactive biochemically and, with field
strains particularly they have many reactions in common.

Tests for S. hyodysenteriae antigens

• The fluorescent antibody test is useful as a screening test foriden tification of S. hyodysenteriae

• A slide agglutination test and a microscopic agglutination lest using absorbed polyclonal serum
 153

have been described.

Tests for S. hyodysenteriae antibodies

Several serological procedures have been developed for the serodiagnos is of swine
dysentery and for the detection of carrier animals. The ELISA appears to be the most sensitive but
should be used as a herd test, rather than for individual animals, as false-positive and false-negative
results can occur.

BORRELIA

Borreliae, which are longer and wider than other spirochaetes, have a similar helical shape. In
addition to a linear chromosome, which is unique among bacteria, borreliae possess linear and
circular plasmids. Although these spirochaetes can cause disease in animals and humans, subclinical
infections are also common. Borreliae are transmitted by arthropod vectors.

USUAL HABITAT

Borreliae are obligate parasites in a variety of vertebrate hosts. Although these organisms
persist in the environment for short periods, they depend on vertebrate reservoir hosts and arthropod
vectors for long-term survival.

DIFFERENTIATION OF BORRELIA SPECIES

Borreliae can be differentiated from other spirochaetes by their morphology, by the low
guanine and cytosine content of their genomic DNA and by ecological, cultural and biochemical
characteristics. Identification of Borrelia species depends mainly on genetic analysis. At least nine
genospecies or genomic groups of B.burgdorferi sensulato, have been identified using DNA-DNA
hybridization, 16s rRNA sequencing and other molecular techniques.

CLINICAL INFECTIONS

The species of particular veterinary importance are B. burgdorferi sensu lato, the cause of
Lyme disease in animals and humans, and B. anserina which causes avian borreliosis. The
significance of two other species, B.theileri and B.coriaceae, as animal pathogens, is uncertain.

LYME DISEASE

This condition, also known as Lyme borreliosis, was first identified in 1975 following
investigation of a cluster of arthritis cases in children near the town of Old Lyme, Connecticut. The
causative agent, a spirochaete, was named Borrelia burgdorferi.

Epidemiology

Lyme disease has been reported in humans, dogs, horses and cattle, and infection has been
documented in sheep. Ticks are the only competent vectors of B. burgdorferi sensu lato. Infection is
usually acquired by larval stages of ticks feeding on small rodents.

The most common tick vector for B. burgdorferi sensu lato in Europe is Ixodes ricinus; in
central and eastern USA it is I. scapularis; on the west coast of the USA, it is I. pacificus, and in
Eurasia it is I. persulcatus. Transovarial transmission of the spirochaete in the tick, which may occur
infrequently, is not epidemiologically important. Occasional transmission of borreliae from infected
incidental hosts to uninfected ticks may occur. Although B. burgdorferi sensulato has been
demonstrated in the urine of dogs and horses, infected urine is an unlikely source of infection.
 154

Pathogenesis

Transmission of B. burgdorferi sensulato occurs when an infected tick feeds on a susceptible

animal. Prior to feeding, the spirochaetes are restricted to the midgut of the ticks and, following
ingestion of blood, they are found in the salivary glands. Following ingestion of blood by the tick, a
change occurs in the expression of the outer surface protein (Osp) of the borreliae. This change in
Osp expression from OspA to OspC appears to be essential for virulence.

After entering the bloodstream of a susceptible host, borreliae multiply and are disseminated
throughout the body. Organisms may be demonstrated in joints, brain, nerves, eyes and heart.
Whether disease is caused by active infection or by host immune responses to the organism is
unclear. Recent studies suggest that persistent infection, leading to the induction of cytokines,
contributes to the development of lesions. There may be an association between different genotypes
of B.burgdorferi and particular clinical syndromes in humans.

Clinical signs

Most infections are subclinical. Serological surveys demonstrate that exposure is common in
both animal and human populations in endemic areas. The clinical manifestations of Lyme disease
relate mainly to the sites of localization of the organisms. Clinical disease is reported frequently in
dogs. Signs include fever, lethargy, arthritis and evidence of cardiac, renal or neurological disturbance.
The clinical signs in horses are similar to those in dogs and include lameness, uveitis, nephritis,
hepatitis and encephalitis. Lameness in cattle and sheep associated with B.burgdorferi sensu lato
infection has been reported.

Diagnosis

Laboratory confirmation of Lyme disease may prove difficult because the spirochaetes may
be present in low numbers in specimens from clinically affected animals. In addition, the organism is
fastidious in its cultural requirements.

A history of exposure to tick infestation in an endemic area in association with characteristic

clinical signs may suggest Lyme disease. The ELISA is extensively used for antibody detection;
Western immunoblotting is sometimes used for confirmation of ELISA results. Immunofluorescence
assays may also be used but, in common with ELISA, the results of these methods may be difficult to
interpret.

Culture of borreliae from clinically affected animals is confirmatory. Cultures in Barbour-

Stoenner-Kelly medium should be incubated for six weeks under microaerophilic conditions and
should be carried out in specialized laboratories. Low numbers of borreliae can be detected in
samples by PCR techniques. These techniques can also be used for identifying genospecies and for
epidemiological investigations.

Treatment and control

Acute Lyme disease responds to treatment with amoxycillin and oxytetracycline. In chronic
disease prolonged or repeated courses of treatment may be required. Acaricidal sprays, baths or dips
should be used to control tick infestation. Where feasible, tick habitats such as rough brush and scrub
should be cleared. Prompt removal of ticks from companion animals may prevent infection. However,
because some tick species can transmit spirochaetes shortly after attachment, it cannot be assumed
that daily removal of ticks will prevent infection. A number of vaccines, including whole cell bacterins
and a recombinant subunit vaccine, are commercially available for use in dogs.

AVIAN SPIROCHAETOSIS
 155

This acute disease of birds, caused by Borrelia anserina, can result in significant economic
loss in flocks in tropical and subtropical regions where the disease is endemic. Chickens, turkeys,
pheasants, ducks and geese are susceptible to infection. Soft ticks of the genus Argas frequently
transmit the disease. However, when there is contact between susceptible birds and infected material
such as blood, tissues or excreta, transmission may occur. Because B.anserina survives poorly in the
environment and for a limited time in infected birds, Argas ticks are important reservoirs of the
organisms.

The borreliae survive trans-stadial moulting in ticks and can be transmitted transovarially
between tick generations. Outbreaks of avian spirochaetosis coincide with periods of peak tick
activity during warm, humid seasons. Morbidity and mortality are low in flocks continually exposed to
infection. The disease is characterized by fever, marked anaemia and weight loss. Paralysis may
develop as the disease progresses. Immunity, which follows recovery, is serotype specific. Several
serotypes may be present in a particular region.

Diagnosis can be confirmed by demonstration of the spirochaetes in buffy coat smears using
dark-field microscopy. Blood or tissue smears can also be examined using immunofluorescence.
Giemsa-stained smears or silver impregnation techniques can be used to demonstrate the borreliae in
tissues. The organisms are usually isolated by inoculating embryonated eggs or young chicks with
infected blood or homogenized tissues.

Treatment with antibiotics is effective. Inactivated vaccines and tick eradication are the main
control measures.

BRACHYSPIRA

Five genospecies of intestinal spirochaetes have been isolated from pigs namely Brachyspira
hyodysenteriae, B. pilosicoli, B. innocens, Serpulina intermedia and S. murdochii. The genera Serpulina
and Brachyspira were recently combined. These anaerobic spirochaetes have six to fourteen spirals
and are 0.1 to 0.5 µm in width.
 156

USUAL HABITAT

Pathogenic Brachyspira species are found in the intestinal tract of both clinically affected and
normal pigs. Carrier pigs can shed B.hyodysenteriae for up to three months and are the principal
source of infection for healthy pigs.

DIFFERENTIATION OF BRACHYSPIRA SPECIES

The differentiation of B.hyodysenteriae from other intestinal spirochaetes is based on its

pattern of haemolysis on blood agar. Tests for detecting indole production or the hydrolysis of
hippurate are also useful diagnostically. Restriction endonnclease analysis, restriction fragment
length polymorphism, ribotyping using 16s rRNA analysis, PCR-based assays and multilocus enzyme
electrophoresis have been developed both for differentiating species and for distinguishing strains of
organisms within species. Brachyspira hyodysenteriae isolates can also he allocated to several
serogroups and serotypes.

PATHOGENESIS

Most information on the pathogenesis of Brachyspira species derives from studies of

B.hyodysenteriae. Motility in mucus is an essential virulence factor of this organism; mutant strains
with altered motility are less capable of colonizing the pig intestine. Colonization may be enhanced by
factors in mucus with chemotactic activity for the organisms. Factors with such chemotactic
activities have been demonstrated in vitro. Haemolytic activity, demonstrated in vitro, correlates with
pathogenicity and three genes encoding haemolytic and cytotoxic activity have been cloned and
sequenced.

The pathogenesis of infection with B.pilosicoli differs from that of B. hyodysenteriae in that
attachment of the spirochaetes to the intestinal mucosa appears to be important. Attachment of
B.pilosicoli to the epithelial cells of the colonic mucosa leads to disruption of function with resultant
cell shedding and oedema.

CLINICAL INFECTIONS

Infections with Brachyspira species are of importance in pigs. Brachyspira hyodysenteriae,

the cause of swine dysentery, and B. pilosicoli, the cause of porcine intestinal spirochaetosis, are
recognized pathogens. There is evidence that Serpulina intermedia may be associated with porcine
spirochaetal colitis, but this has not been confirmed experimentally. Pigs acquire infection through
exposure to contaminated faeces. The disease usually spreads slowly through a herd, affecting only
one or two pens at a time. Dogs, rats, mice and flies may act as transport hosts for the spirochaetes.
Mice populations can maintain B. hyodysenteriae. Although strains of B. pilosicoli have been found in
many species including humans, dogs, chickens and pheasants, cross-infection between species has
not been clearly demonstrated. Brachyspira species can survive in the environment for limited periods
only if protected from desiccation. Brachyspira hyodysenteriae can persist for several weeks in moist
faeces and for at least three days in slurry.

CLINICAL SIGNS

Infection with B. hyodysenteriae causes dysentery which is most often encountered in

weaned pigs from six to twelve weeks of age. Affected pigs lose condition and become emaciated.
Appetite is decreased and thirst may be evident. During recovery, there may be large amounts of
mucus in the faeces. Although mortality is low, reduced weight gains due to poor food conversion
cause major economic loss.

Brachyspira pilosicoli was identified in 1996 as the cause of porcine intestinal spirochaetosis.
Previously, enteric disease had been produced experimentally by infecting pigs with a weakly-
 157

haemolytic spirochaete. The clinical signs in porcine intestinal spirocbaetosis are similar to those of
swine dysentery but are less severe. Diarrhoea contains mucus rather than blood. Reduced feed
conversion efficiency with poor weight gain has a major effect on production.

DIAGNOSIS

History, clinical signs and gross lesions may indicate swine dysentery. Blood agar with added
antibiotics is used for the culture of Brachyspira species. Cultures are incubated anaerobically at 42°C
for at least three days. Complete haemolysis is present around colonies of B. hyodysenteriae; other
enteric spirochaetes are weakly haemolytic. Definitive identification can be made using
immnnofluorescence, DNA probes or biochemical tests. Serological tests such as ELISA can be used
to investigate infection in herds. PCR-based techniques have been developed and may be useful for
laboratory confirmation.

TREATMENT AND CONTROL

Medication of drinking water is a useful method of treatment. Drugs commonly used include
tiamulin, lincomycin and the nitroimidazoles. Improved hygiene, medication of feed and alteration of
the diet may assist in controlling infection. Depopulation, thorough cleaning and disinfection of
premises and strict rodent control are required for eradication of the disease.

MYCOPLASMA

The mycoplasmas are microorganisms in the class Mollicutes. Of the nine genera in this
class, five contain species of veterinary interest. The genus Mycoplasma, in which there are about 100
species, contains most of the animal pathogens. The first mycoplasma identified in 1890 was
Mycoplasma mycoides subspecies mycoides, the cause of contagious bovine pleuropneumonia.
Similar types of mycoplasmas which were subsequently identified were called pleuropneumonia- like
organisms (PPLO).

Mycoplasmas, the smallest prokaryotic cells capable of self-replication, are pleomorphic

organisms ranging from spherical (0.3 to 0.9 pm in diameter) to filamentous (up to 1.0 µm long).
Because they cannot synthesize peptidoglycan or its precursors, they do not possess rigid cell walls
but have flexible, triple-layged outer membranes.

Their flexibility allows them to pass through bacterial membrane filters of pore size 0.22 to
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0.45 µm. Mycoplasmas are susceptible to desiccation, heat, detergents and disinfectants. However,
they are resistant to antibiotics such as penicillin which interfere with the synthesis of bacterial cell
walls. Based on 5s rRNA sequence analyses, the mycoplasmas have been shown to be linked
phylogenetically to Gram-positive bacteria such as Clostridium species which have low guanine-
cytosine content in their DNA. They require enriched media for growth, characteristically forming
umbonate micro-colonies when illuminated obliquely and microcolonies with a 'fried egg' dppearance
in transmitted light. The dense central zone is due to extension of the microcolony into the agar.
Mycoplasmas, which have relatively small genomes (approximately 800 genes) are fastidious in their
growth requirements.

Most mycoplasmas are facultative anaerobes and some grow optimally in an atmosphere of
5 to 10% CO2. Nonpathogenic anaerobic mycoplasmas are found in the rumens of sheep and cattle.
The genera Mycoplasma and Ureaplasma contain animal pathogens.

USUAL HABITAT

Mycoplasmas are found on mucosal surfaces of the conjunctiva, nasal cavity, oropharynx and
intestinal and genital tracts of animals and humans. Some species have tropisms for particular
anatomical sites while others are found in many locations. In general, they are host-specific and
suwive for short periods in the environment.

COLONIAL MORPHOLOGY

When examined microscopically at low magnification, unstained microcolonies of

Mycoplasma species are 0.1 to 0.6 mm in diameter and have a 'fried-egg' appearance. Some species
produce colonies up to 1.5 mm in diameter which can be seen without magnification.

Colonies of Ureaplasma species are usually 0.02 to 0.06 mm in diameter and often lack a
typical peripheral zone. Because their colonies are tiny, these organisms were formerly referred to as
T-mycoplasmas.

Dienes stain facilitates recognition of microcolonies by staining the central zone dark blue
and the peripheral zone a lighter blue.

Microcolonies of Mycoplasma species require differentiation from colonies of bacterial L-

forms. However, L-forms often revert to normal and produce cell walls and typical bacterial colonies
when subcultured on non-inhibitory media.

Mycoplasma species and Ureaplasma species require sterols for growth, and this is reflected
in their sensitivity to inhibition by digitonin. As Acholeplasma species are sterol-independent, they are
resistant to inhibition by digitonin. In the digitonin sensitivity test, a filter paper disc impregnated with
digitonin is placed on medium inoculated with the isolate. A zone of growth inhibition exceeding 5
mm around the disc indicates sensitivity to digitonin.

PATHOGENESIS AND PATHOGENICITY

Mycoplasmas adhere to host cells, an attribute essential for pathogenicity. This close contact
facilitates toxic damage to the host cells by soluble factors produced by the pathogen. Some
pathogenic species possess structures composed of unique adhesion proteins which promote
attachment to mammalian cells. Mycoplasmas can adhere to neutrophils and macrophages and can
also impair phagocytic functions. In addition, individual species damage cells by active penetration.

Modulation or activation of host immune responses is critical in the pathogenesis of

mycoplasmal diseases. Some pathogenic mycoplasmas, including those involved in pulmonary
diseases, are mitogenic for B and T lymphocytes. Activation of macrophages and monocytes leads to
 159

the release of cytokines including tumour necrosis factor and interleukins, resulting in the initiation of
inflammation.

Pneumonia-producing mycoplasmas, which adhere to ciliated respiratory epithelium, can

induce ciliostasis, loss of cilia and cytopathic change. Inflammation can also be induced in the bovine
mammary gland by a membrane associated toxin of M. bovis.

DIAGNOSTIC PROCEDURES

The presence of Mycoplasma species or mycoplasmal antigens in samples can be

demonstrated immunologically or by nucleic acid procedures:

 Fluorescent antibody techniques

 Peroxidase-antiperoxidase procedures on paraffin embedded tissues

 Polymerase chain reaction techniques

 Inoculated mycoplasmal medium is incubated, aerobically or in 10% CO2, in a humid

atmosphere at 37°C for up to 14 days.

 Fluid samples can be inoculated directly onto agar or into broth media. Tissue specimens
such as lung should he freshly sampled and a cut surface moved across the surface of a
solid medium. Alternatively, the tissue can be homogenized in broth and samples of the
suspension used for inoculation of liquid or solid media.

Identification criteria for isolates:

- 'Fried-egg' microcolonies

- Microcolony size

- Cholesterol requirement for growth (digitonin sensitivity test)

- Biochemical profile including urease production

- Fluorescent antibody technique on microcolonies

- Growth inhibition test with specific antisera

Serological tests:

- Complement fixation tests for the major mycoplasma1 diseases of ruminants are used for
certification when animals are traded internationally.

- Tests based on ELISA are being developed for the diagnosis of economically important
mycoplasmal diseases.

- Rapid plate agglutination tests are used for screening poultry flocks and for the field
diagnosis of contagious bovine pleuropneumonia.

- Haemagglutination-inhibition tests can be used to determine the antibody levels in avian

mycoplasmal diseases.

CLINICAL INFECTIONS

Mycoplasmas are often involved in disease Erocesses affecting mucosal surfaces. Factors
 160

such as extremes of age, stress and intercurrent infection may predispose to tissue invasion. In
addition, mycoplasmas may exacerbate disease initiated by other pathogens, particularly in the
respiratory tract.

Mycoplasmal infections cause respiratory diseases of major economic importance in farm

animals especially in ruminants, pigs and poultry. Infections associated with mastitis or conjunctivitis
in cattle and with disease conditions in domestic carnivores are usually of lesser importance. Several
mycoplasmas have been isolated from dogs and cats but their precise role in disease has not been
clearly defined. They have been implicated in respiratory and urinary tract disease in dogs. In cats, M.
felis can occasionally cause conjunctivitis and M.gateae is associated with arthritis.

CONTAGIOUS BOVINE PLEUROPNEUMONIA

Contagious bovine pleuropneumonia (CBPP) is a severe contagious disease of cattle which

has been recognized for more than 200 years and formerly had a worldwide distribution. It is caused
by M. mycoides subspecies mycoides (small colony type), a member of the 'mycoides cluster'. This
cluster is composed of six closely related members including the M. mycoides and M. capricolwn
subspecies of sheep and goats and bovine mycoplasma group 7. Members of the cluster share
biochemical, immunological and genetic characteristics which render individual species and
subspecies difficult to differentiate.

The main method of transmission is by aerosols. Transmission of the disease requires close
contact with clinically affected animals or asymptomatic carriers. Clinical signs become apparent
three weeks after infection. The severity of the disease relates to strain virulence and the immune
status of the host. Spread of infection can be relatively slow with peak morbidity (about 50%) at 7 to 8
months after introduction of infection into a herd. In severe outbreaks the mortality rate may be high.

Clinical signs and pathology

Clinical signs in the acute form of CBPP include sudden onset of high fever, anorexia,
depression, drop in milk yield, accelerated respiration and coughing. Animals adopt a characteristic
stance with the head and neck extended and elbows abducted. Expiratory grunting and mucopurulent
nasal discharge may be present. Death can occur 1 to 3 weeks after the onset of clinical signs.

Arthritis, synovitis and endocarditis may be present in affected calves. At postmortem, the
pneumonic lungs have a marbled appearance. Grey and red consolidated lobules alternate irregularly
with pink emphysematous lobules and the interlobular septa are distended and oedematous. There
may be abundant serofibrinous exudate in the pleural cavity. In chronic cases, fibrous encapsulation
of necrotic foci is commonly found. These necrotic foci contain viable mycoplasmas and breakdown
of the capsules in chronically affected animals is a major factor in the persistence and spread of
CBPP in endemic areas.

Diagnosis

In endemic regions, clinical signs and characteristic postmortem findings allow a

presumptive diagnosis. Techniques, such as the polymerase chain reaction, based on the detection of
specific DNA in tissue samples can be used to differentiate M. Mycoides subspecies mycoides (small
colony type) from other members of the 'mycoides cluster'. The fluorescent antibody test can be used
on pleural fluid to confirm the presence of the pathogen. Isolation and definitive identification of the
pathogen from broncho-alveolar lavage, pleural fluid, lung tissue or the broncho-pulmonary lymph
nodes is confirmatory. Polymerase chain reaction-based tests may be useful confirmatory tests.
 161

Serological tests:

- Rapid field serum agglutination test

- Passive haemagglutination screening test

- Complement fixation test for determining disease status of animals crossing national
boundaries

- Dot-blot technique for confirmation

- A competitive ELlSA is presently under development.

TREATMENT AND CONTROL

Although treatment with antimicrobial drugs may be attempted in countries where the
disease is endemic, it is generally unsatisfactory especially for chronically affected animals. In
countries where CBPP is exotic, slaughter of affected and in-contact cattle is mandatory. In endemic
regions, control strategies are based on prohibiting movement of suspect animals, mandatory
quarantine and the elimination of carrier animals by serological testing and slaughter.

Annual vaccination with attenuated vaccines is carried out to stimulate effective immunity in
cattle in endemic areas. The virulence of attenuated vaccines varies with the strain of mycoplasma
employed. Annual vaccination may be discontinued as eradication of the disease progresses.

INFECTIONS WITH MYCOPLASMA BOVIS

Strains of M.bovis, which is worldwide in distribution, can cause severe pneumonia in calves
in the absence of other respiratory pathogens and can exacerbate respiratory disease caused by
Pasteurella and Mannheimia species. Mycoplasma bovis has also been associated with mastitis and
polyarthritis.

Diagnostic techniques are similar to those used for other mycoplasmas. A number of other
Mycoplasma species cause sporadic cases of mastitis in cattle. Although the mastitis may be severe,
systemic involvement is uncommon. There is often a dramatic loss of milk production and the serous
or purulent mastitic exudate has a high leukocyte count. Mycoplasmal mastitis should be considered
when other common bacterial causal agents have been excluded.

CONTAGIOUS AGALACTIA OF SHEEP AND GOATS

This severe febrile disease of sheep and goats, caused by M.agalactiae, is prevalent in parts
of Europe, northern Africa and parts of Asia. It usually becomes evident immediately after parturition
and is characterized by mastitis, arthritis and conjunctivitis. Pregnant animals may abort and the
disease can be fatal in young animals due to pneumonic complications. The organism is shed in milk
and may remain localized in the supramammary lymph nodes between lactations. Disease due to M.
agalactiae must be distinguished from mastitis and arthritis associated with M. capricolum
snbspecies capricolum, M. mycoides subspecies mycoides (large colony type) and M. mycoides
subspecies capri. Inactivated and attenuated vaccines for M.agalactiae are commercially available.

CONTAGIOUS CAPRINE PLEUROPNEUMONIA

Contagious caprine pleuropneumonia (CCPP), caused by M. capricolum subspecies

capripneumoniae (formerly Mycoplasma strain F38), is present in northern and eastern Africa and in
Turkey. The disease is characterized by pneumonia, fibrinous pleurisy, profuse pleural exudate and a
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marbled appearance on the cut surface of affected lungs.

Although similar in many respects to CBPP, well developed necrotic areas in the lungs in
chronic CCPP are rare. The disease is highly contagious and is transmitted by aerosols. Nomadic
herds often carry infection to regions free of the disease. Pleuropneumonia in goats can occasionally
be caused by M.mycoides subspecies capri or M.mycoides subspecies mycoides (large colony type).
However, monoclonal antibody to M.capricolum subspecies capripneumoniae is specific for this
organism in a growth inhibition disc test. Inactivated vaccines give satisfactory protection.

MYCOPLASMAL DISEASES OF POULTRY

Mycoplasma gallisepticum causes chronic respiratory disease in chickens and infectious

sinusitis in turkeys. The organism is transmitted through infection of the embryo in the egg or by
aerosols. Clinical signs are consistent with upper respiratory tract involvement in chickens. In turkeys,
there is swelling of the paranasal sinuses.

Reduced egg production may be evident. Diagnosis is based on isolation and identification of
the pathogen and on flock testing using the serum plate agglutination test. Haemagglutination
inhibition and ELISA tests are also used in flocks to confirm infection. Although antimicrobial
medication of feed is used during outbreaks, the establishment of specific-pathogen-free flocks is the
preferred method for controlling the disease. Eggs used for hatching should be dipped in a tylosin
solution to eliminate the pathogen. Modified live vaccines and bacterins are available.

Mycoplasma meleagridis may be egg-transmitted and may be present in turkey semen.

Aerosol transmission is less important with this pathogen than with M. gallisepticum.

The clinical features of the infection include reduced egg hatchability, airsacculitis in young
poults and joint and bone deformities in growers. Confirmation requires isolation and identification of
the pathogen. The serum plate agglutination test is used for flock testing. Tylosin, administered in the
water for the first 10 days of life is of therapeutic value. Eggs used for hatching should be dipped in
tylosin solution. Semen should be obtained from M. meleagridis-free toms.

Mycoplasma synoviae, the cause of infectious synovitis in chickens and turkeys, is

transmitted mainly by aerosols. Egg transmission is much less important than in M. gallisepticum and
M.meleagridis infections. Synovitis, arthritis and respiratory signs are the main clinical features.

Confirmation requires isolation and identification of the pathogen or positive serological tests.
Tetracycline medication of the feed is used for treatment and control. Eradication is possible through
the development of specific-pathogen-free flocks.
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COXIELLA BURNETII

INTRODUCTION

Q fever is a zoonotic bacterial infection caused by Coxiella burnetii, an obligate intracellular

parasite, classified within the family Rickettsiaccae. Q fever (Q for “query”) was first used in 1937 to
describe a mysterious febrile illness of packing house workers in Brisbane, Australia. The causative
agent was isolated from infected workers and later was identified as Rickettsiae. Almost
simultaneously the same organism was identified wood tick collected in Montana. Cox, an American,
and Burnet, an Australian, where honored for their early work with this organism, hence the name
“Coxiella burnetii”.

HOST AND SUSCEPTIBILITY

Coxiella burnetii is a well-established infectious agent that has reached a state of balanced
pathogenicity in a plethora of host. Human are unnatural and usually dead-end-host. The
microorganism generally maintained in a less pathogenic form in separate cycle existing
independently among wild mammals and their haematophagus arthropods, principally tick and in
domestic animals.

MORPHOLOGY

It is an intracellular organism which is a polymorphic bacillus (0.2-0.4mm width, 0.4-1.0 mm

length), which has a cell membrane, like Gram negative bacteria. However, it stains poorly with
pigmented Gram stain, but gimenez staining is traditionally used to stain the Coxiella burnetii
pathogen from pathological materials and crop.

Coxiella burnetii has several distinctive characteristics, including a sporulation-like process

that protects the organism against the external environment, where it can survive for long periods. In
mammals, the usual host cell of C. burnetii is the macrophage, which is unable to kill the bacterium.
The other an important characteristic of C. burnetii is its antigenic variations, those called phase
variation. This antigenic shift can be measured and is valuable for differentiating acute from chronic Q
fever. Thus, it displays two antigenic phases those are phase-I and phase- II that are liable to the
Lipopolysaccharide (LPS) of the membrane.

Phase I Coxiella burnetii antigens are corresponds to the smooth phase of Gram-negative
bacteria and are more highly infectious and phase-II antigen is corresponding to the granular (Rough)
phase which has a lower virulence. The strain of Coixella burnetii pathogens are grouped into six (I VI)
 164

genomic group based on the Restriction Fragment Length Polymorphism (RFLP). The pathogenicity
and the virulence of the C. burnetii are associated with genetic characteristics, type of strains and
plasmid groups and with host factors such as pregnancy.

Characteristics:

 Small, Gram-negative, pleomorphic coccobacillus; obligate intracellular bacterium that

replicates in macrophages and monocytes.

 Order: Legionellales; Family: Coxiellaceae

 Size: 0.4-1.2 mm in length and 0.2-0.4 mm in width

 Nucleic acid: Coxiella genome is approximately 2000 kb.

PHYSICOCHEMICAL PROPERTIES

Resistant to heat, low or high pH, 0.5% sodium hypochlorite, UV irradiation, and environmental
conditions such as desiccation, extreme temperatures and sunlight because of the presence of a
spore-like stage. Reported to survive for 7-10 months on wool at 15-20°C, for more than 1 month on
fresh meat in cold storage, and for 40 months in skim milk at room temperature.

The microorganism has two antigenic forms: phase I and phase II. Phase I is the highly
infectious form found in nature and has intact lipopolysaccharide (LPS) on the cell membrane,
whereas phase II is laboratory-grown, attenuated, avirulent in animals, and has truncated LPS.

Infected cells contain 2 structural forms of the bacteria: large cell variant (LCV) or vegetative
forms, and small cell variant (SCV) or condensed forms. SCV released during lysis of infected cells
result in the spore-like form found in the environment.

Coxiella burnetii does not grow in artificial media, but can be cultured in embryonated eggs,
primary cells, several cell lines including Vero cells and a number of macrophage-like cell lines.

Stain red with modified Ziehl Nelson and Machiavello procedures, and purple with Giemsa
stain. They are gram negative, non-motile and varies in shape from pleomorphic to ovoid even to rod
like.

On adaptation to laboratory culture, Coxiella burnetii undergoes modification of antigenicity

and virulence from phase I (analogous to smooth gram negative bacteria) found in animals to phase II
(rough from). However, this variation of antigenicity has relevance for vaccine formation and
serological diagnosis.

Phase variation:

Coxiella burnetii has two antigenic surface phases; phase I and phase II, which vary in their
pathogenic and immunogenic properties and undergo antigenic phase variation due no
Lipopolysaccharide truncation when serially passaged in embryonated eggs or cell culture. The phase
I form is extremely infectious and exists in human and other animals. Passaging can also result in a
shift to form spore which allows the organism to survive in harsh environment. Indeed, it can survive
for greater than 40 months in skim milk at room temperature and is readily recovered from soil up to
one month after contamination. It has high level of resistance to chemical and physical agents. High
level of resistance to heat has been attributed to the formation of endospore.

DISINFECTION

C. burnetii is relatively resistant to disinfectants. The infective dose is also reported to be low.
 165

Agents reported to be effective with a contact time of 30 minutes include 70% ethanol and some
quaternary ammonium-based disinfectants (e.g., MicroChem-Plus®, 5% Enviro-Chem®). This
organism can also be inactivated with 5% hydrogen peroxide, or by gaseous formaldehyde, 5%
chloroform or ethylene gas in a sealed, humidified chamber. Variable susceptibility has been reported
for hypochlorite, phenolic disinfectants and formalin.

®
Sodium hypochlorite (1:100 dilution of household bleach) or 1% Virkon S result in greater
than 90% reduction in infectivity. Although 2% formaldehyde is reported to destroy C. burnetii, it has
been isolated from tissues stored in formaldehyde for several months. Sources also differ on the
effectiveness of Lysol®, which has changed its formulation a few times. Physical inactivation can be
accomplished by gamma irradiation or high heat, including high temperature pasteurization of milk
(e.g., 161°F/72°C for 15 seconds).

SOURCE OF INFECTION AND TRANSMISSION

Infection of non-pregnant animals is clinically silent and is followed by latent infection until
pregnancy when there is recrudescence with infection in the intestine, uterus, placenta and udder, and
excretion from this site at parturition.

The organism is present in high concentration in the placenta and fetal fluids, and subsequent
vaginal fluids. It is also excreted in urine, and through milk. Cattle and small ruminants can shade the
organism through faeces, sperm and reproductive discharge. Parturient cats and dogs have also been
implicated as source of human infection.

Transmission is by direct contact and through inhalation. There is significant contamination

of the environment of infected animals at the time of parturition and this is probably a critical period
for transmission of the disease within herds and flocks.

Coxiella burnetii is transmitted from various reservoir hosts to human through direct contact
as well as air born, vector born, and through contaminated vehicle. In ticks, there is transovarian
(transmission of Coxiella burnetii from adult tick to egg) and transstadial (transmission of Coxiella
burnetii from larvae to nymph, and adult). The organism is present in the semen of seropositive bulls
and venereal transmission is suspected.

Many arthropods including cockroaches, beetles, flies, fleas, bugs, lice, mite and ticks are
naturally or experimentally infected; human rarely acquired the disease by bites of arthropods. Most
human cases of Q fever can be traced to exposure to infected sheep, cattle and goats either directly
or through unpasteurized milk.

PATHOGENESIS AND PATHOGENICITY

The organism probably follows the oropharyngeal route as its port of entry into the lungs and
intestines of both humans and animals. It is highly infectious, and a very low dose is sufficient to
initiate infection. Primary multiplication takes place in the regional lymph nodes after the initial entry,
and a transient bacteraemia develops which persists for five to seven days.

Coxiellaburnetii has two morphologically distinct cell variants; an intracellular and

metabolically active large cell variant (LCV) and a spore like small cell variant (SCV). These two forms
are morphologically and functionally distinct. The LCV is larger, elongated less electron-dense
bacteria and metabolically active and replicating large bacteria. While, the SCV presents a compact
rod-shaped with a very dense central region and it is considered the metabolically dormant and less
replicating. The SCV are shed by infected animals.

After infection the organism attaches to the cell membrane of phagocytic cell. After
phagocytosis, the phagosome containing the SCV fuses with the lysosome. The SCV are
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metabolically activated in the acidic phagolysosomes and can undergo vegetative growth to form LCV.

The LCV and the activated SCV can both divide by binary fission and the LCV can also
undergo sporogenic differentiation. The spores that are produced can undergo further development to
become metabolically inactive SCV. And both spores and SCV can then be released from the infected
host cell by either cell lysis or exocytosis.

The acidic environment also protects Coxiella burnetii from the effects of antibiotics, as the
efficacy of antibiotics is decreased in the acidic PH. The SCV and spore forms are more difficult to
denature than LCV.

Coxiella burnetii also has two distinct antigenic phases, Phase I and Phase II, based on
changes that occur in the organism during in vitro culture. The primary significance of these two
phases is that antibodies to phase II antigens are made during the early stages of the infection, but
antibodies to phaseI antigens predominate if the organism persists longer. This switch is used to
distinguish acute from chronic infections in people, although it is not currently employed in animals.
Phase I bacteria (wild virulent type) with a smooth full length LPS were isolated from infected humans,
animals and arthropods. Phase I bacterium converts to an avirulent phase II with rough LPS after
several passages in embryonated egg or cell cultures. The virulence and the pathogenicity of the
Coxiella burnetiiare associated with genetic characteristics, plasmid groups and type of strains and
also with host factors such as pregnancy.

After Coxiella burnetii enter to the host and gain access to vascular endothelium and
respiratory and renal epithelium, it multiplies in phagosomes; due to an enzyme system adapted low
pH (5.0). It causes necrosis and hemorrhage of many other organs including the liver, central nervous
system and mononuclear phagocytic system. In animals the pattern may be similar, although mildly
affected animals may have latent infection. Latent infection can persist particularly in the locating
mammary gland and the pregnant uterus but become reactivated during parturition. Abortion
(sporadically) may result from placentitis or the delivery may be normal and produce viable young.
Immune complex pneumonia can develop in many organs.

CLINICAL SIGNS

In ruminants, significant clinical signs seem to be limited to pregnant animals, and are
characterized by abortions, stillbirths, and the birth of small or weak offspring. Reproductive losses
may occur as outbreaks in sheep and goats, but they seem to be sporadic in cattle. Most abortions
are reported to occur near term. Anorexia, depression, agalactia and retained fetal membranes are
possible, but they seem to be uncommon, and most abortions have no significant premonitory signs.
Subsequent pregnancies might sometimes be affected. Links between infection with C. burnetii and
endometritis/ metritis or infertility have been suggested in cattle and sheep, and a possible link with
subclinical mastitis has been proposed in cattle.

POST MORTEM LESIONS

C. burnetii abortions in ruminants are characterized by placentitis primarily affecting the

intercotyledonary areas. The placenta is typically leathery and thickened, and it may contain large
amounts of mucopurulent or purulent exudates, especially at the edges of the cotyledons and in the
intercotyledonary areas. Severe vasculitis is uncommon, but thrombi and some degree of vascular
inflammation may be noted. Aborted fetuses tend to be fresh, though they are occasionally autolyzed.
Fetal lesions are usually non-specific, although pneumonia and microscopic evidence of hepatic
necrosis or granulomatous inflammation have been reported.

DIAGNOSIS

Collection of Samples
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In the acute-phase of the disease, immediately serum sample should be collected after the
onset of symptoms (within the first 2 weeks) with a convalescent-phase specimen collected 3–6
weeks later. Before antibiotic administration the whole blood should be collected in EDTA-treated
anticoagulant tubes and shipped refrigerated on the frozen gel packs by overnight. The Buffy coat can
be saved for DNA amplification and stored frozen in a non–frost-free freezer if samples are to be
prepared for other laboratory tests. The most commonly evaluated sample used for confirmation for
chronic Q fever is heart valve tissue.

Staining and Related Measures

The diagnosis of coxiellosis in aborted small ruminants by an ordinary method is to detect the
pathogen using staining techniques. The smears prepared from the samples are usually stained by
Gimenez, Stamp, Giemsa stain or Machiavello.

Isolation and Cultivation:

For routine diagnosis of Q fever cultivation of C. burnetii is not recommended, since the
process is very difficult, time consuming, and dangerous. In addition to this culture of C. burnetii
requires a bio-safety level 3 (BSL- 3) laboratory because bacteria are highly infective and can be
hazardous for laboratory workers. Patients with chronic Q fever have already received antibiotics
which can further complicate isolation attempts; a negative culture does not rule out a C. burnetii
infection. Specimens can be referred to CDC through state public health laboratories for culture.

Isolation must be made in cell culture of macrophage or fibroblast lines, embryonated eggs or
laboratory rodents, but cell dissociated to phase II because phase I is highly infectious.

Phase I antigens are isolates from organisms taken directly from animals or their patients.
This natural phase is highly infectious, contain large amount of lipopolysaccharides (LPs), and form
smooth colonies in culture. Phase II antigens are round in organisms that have been passed serially in
embryonated eggs, have a truncated, and lack some cell surface antigens.

Because of laboratory acquired infection caused by C. burnetii, cultivation of the organism

has been discouraged. However, the use of a shell vial assay with human lung fibroblast to isolate the
organism from buffy coat and biopsy specimens has not resulted in any laboratory acquired infection.

Isolation of C. burnetii is dangerous to laboratory personnel, requiring BSL 3 conditions, and

culture is rarely used for diagnosis. Embryonated chicken eggs do not work as well as cells, and are
no longer recommended for the initial isolation. Laboratory animals, such as mice and guinea pigs,
have occasionally been employed to isolate C. burnetii, mostly in the past. Various genotyping
methods, such as multiple-locus variable-number tandem repeat analysis (MLVA), multispacer
sequence typing (MST) and single-nucleotide-polymorphisms can be useful for linking outbreaks to
their source.

Serology:

Serological tests including compliment fixation test (CFT), Indirect Fluorescence Antibody
(IFA) and enzyme linked immunosorbent assays (ELISA) and Microagglutination can be used to help
diagnose Q fever.

Molecular Method:

Multispacer Sequence Typing, depending on DNA sequence variations in10 short intergenic
regions, can be performed on isolated C. burnetii strains or directly on extracted DNA from clinical
samples. Polymerase Chain Reaction (PCR) or immuno histochemical methods can be used for
detection of Coxiella burnetii in tissue culture or tissue specimens derived from patients.
 168

Diagnostic laboratories usually use PCR to detect C. burnetii in secretions, excretions and
tissues. Loop-mediated isothermal amplification (LAMP) assays have also been published. Nucleic
acids of C. burnetii can occur in the placenta after a normal delivery, or concurrently with other
pathogens; thus, caution should be used when attributing a clinical case to this organism.
Histopathology and quantitative PCR may be helpful in establishing a causative role. Recently
vaccinated animals can excrete vaccine strains during the first month.

DIFFERENTIAL DIAGNOSIS

In animals the differential diagnosis includes other causes of abortion and infertility like
leptospirosis, brucellosis, Listeriosis, and salmonellosis.

TREATMENTS

Treatment is indicated for all infections, even for those that are subclinical. For domestic
small ruminants’ oral therapeutic dose may be given for 24 weeks.

Doxycycline is the most effective drug against C. burnetii, in most reports. In early studies, the
fluoroquinolones were found to be one of the most effective agents in eliminating C. burnetii from
L929 cells. However, C. burnetii remain susceptible to Levofloxacin, Moxifloxacin, and to a lesser
extent Ciprofloxacin. Erythromycin was proposed as an empirical treatment for C. burnetii pneumonia.
Although C. burnetii is susceptible to a number of antibiotics, as discussed earlier, Oxytetracycline is
the preferred choice.

CONTROL AND PREVENTION

Phase I formalin inactivated Q fever vaccine which provide effective in the immunization of
dairy cattle. Administration of formalin inactivated phase I Coxiella burnetii vaccine in naturally
infected ewes and cows eliminate shedding of the organism in milk. Aborted animals should be
isolated for 3 weeks and aborted and placental contaminated material should be burned. Ideally,
manure should be composted for 6 months before application to fields. Feed areas should be raised
to keep them free from contamination with faeces and urine. Milk and milk products should be
pasteurized.

EHRLICHIA (HEARTWATER – COWDRIOSIS)

AETIOLOGY

Classification of the causative agent

• Ehrlichia ruminantium (formerly Cowdria ruminantium) Order Rickettsiales, Family Anaplasmataceae

• Small, Gram negative, pleomorphic coccus, and obligate intracellular parasite.

• Strains of E. ruminantium are very diverse and vary in virulence: while some strains are highly
virulent, others appear to be less-pathogenic.

• E. ruminantium has a high level of genomic plasticity. Several different genotypes can co-exist in a
geographical area, and may recombine to form new strains.

• E. ruminantium multiplies in vascular endothelial cells throughout the body to cause severe vascular
compromise.

• It usually occurs in clumps of from less than five to several thousand organisms within the
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cytoplasm of infected capillary endothelial cells, and can be detected in brain smears by light
microscopy.

RESISTANCE TO PHYSICAL AND CHEMICAL ACTION

Temperature:

Heat labile and loses its viability within 12–38 hours at room temperature. Infective stabilities
can be cryopreserved in DMSO (dimethyl sulphide) or better yet in sucrosepotassium phosphate-
glutamate medium (SPG). Infective half-life of thawed stabilate kept on ice is only 20–30 minutes.

pH: Not applicable.

Disinfectants: Not applicable.

Survival:

The heartwater organism is extremely fragile and cannot persist outside of a host for more
than a few hours. Because of its fragility, the organism must be stored in dry ice or liquid nitrogen to
preserve its infectivity.

EPIDEMIOLOGY

• Heartwater occurs only where its Amblyomma tick vectors are present.

• Epidemiology depends on interaction of tick vector, causative agent, and vertebrate hosts.

- Tick vector: tick infection rates, seasonal changes influencing abundance and activity, and
intensity of tick control.

- Causative Agent: differing genotypes affecting virulence or stimulation of crossprotection.

- Vertebrate Host: availability of wild animal reservoirs, and age and genetic resistance.

• Because of its extreme fragility, the principal mode of bringing the disease into an area is by
introduction of infected ticks or carrier animals. It is not known for how long wild or domestic
ruminants can be a source of infection for ticks in nature, but it may be many months. Ticks are a
robust reservoir of E. ruminantium, and infection can persist in them for at least 15 months. Careful
dipping and hand-dressing followed by inspection to ensure the absence of ticks is recommended for
animals in transit to heartwater free areas.

HOSTS

• All domestic and wild ruminants can be infected, but the former appear to be the most susceptible.
Indigenous domestic ruminants are usually more resistant to the disease. Wild animals could play a
role as reservoir.

• Heartwater causes severe disease in cattle, sheep, and goats, with milder disease in some
indigenous African breeds of sheep and goats, and inapparent disease in several species of antelope
indigenous to Africa. Bos indicus (Zebu) cattle breeds are in general more resistant than Bos taurus
(European) breeds.

TRANSMISSION

• Heartwater is transmitted transstadially by ticks of the genus Amblyomma, which are biological
vectors of heartwater. Ticks become infected by feeding on acutely ill or subclinically infected
animals. Of the 13 species capable of transmitting the disease, A. variegatum (tropical bont tick) is
 170

by far the most important because it is the most widespread. Other major vector species are the
bont tick A. hebraeum (in southern Africa), A. gemma and A. lepidum (in Somalia, East Africa and
the Sudan).

• A. astrion (mainly feed on buffalo) and A. pomposum (distributed in Angola, Congo and Central
African Republic) are also natural vectors of the disease. Four other African ticks, A. sparsum
(feed on reptiles and buffalo mainly), A. cohaerans (feed on African buffalo), A. marmoreum
(adults occur on tortoises and immature stages on goats) and A. tholloni (adults feed on elephants)
experimentally transmit heartwater.

• Amblyomma ticks are three-host ticks whose life cycles may take from 5 months to 4 years to
complete. Because the ticks may pick up the infection as larvae or nymphs and transmit it as
nymphs or as adults, the infection can persist in the tick for at least 15 months. Infection does not
pass transovarially. While transmission of heartwater can be by adult and nymphal ticks in the field,
in general adults prefer to feed on cattle and nymphs on sheep and goats.

• Cattle egrets have been implicated in the dispersal of Amblyomma ticks in the Caribbean.

• Heartwater can be transmitted vertically and through colostrum of carrier dams.

• Transmission can also occur by intravenous inoculation of blood, tick homogenates or cell culture
material containing E. ruminatium.

SOURCES OF THE AGENT

• Amblyomma ticks fed on an infected vertebrate host.

• Whole blood or plasma of vertebrate host during the febrile reaction, but highest levels of agent
occur during the second or third day of fever.

• Colostrum containing infected cells (reticulo-endothelial cells and macrophages) has been
speculated.

DIAGNOSIS

The average incubation period in natural infections is 2–3 weeks, but can vary from 10 days
to 1 month. The incubation period after intravenous blood inoculation is seven to 10 days in sheep
and goats, and 10 to16 days in cattle. However the incubation is strongly dependent on the dose of
elementary bodies inoculated as shown experimentally using in vitro cultivated E. ruminantium.

Lesions

The gross lesions in cattle, sheep, and goats are very similar. Heartwater derives its name
from one of the prominent lesions observed in the disease, namely pronounced hydropericardium.
The most common macroscopic lesions are hydropericardium, hydrothorax, pulmonary oedema,
intestinal congestion, oedema of the mediastinal and bronchial lymph nodes, petechiae on the
epicardium and endocardium, congestion of the brain, and moderate splenomegaly.

• Accumulation of straw-coloured to reddish fluid in the pericardium is more consistently

observed in sheep and goats than in cattle.

• Hydropericardium, hydrothorax, ascites (mild), mediastinal oedema, and pulmonary oedema

are common and result from increased vascular permeability.
 171

• Oedema of the mediastinal and bronchial lymph nodes may occur.

• Froth in the trachea is often seen, reflecting terminal dyspnoea due to pulmonary oedema.

• Subendocardial petechial haemorrhages are usually present.

• Submucosal and subserosal haemorrhages may occur elsewhere in the body.

• Gross brain lesions are usually absent except for subtle swelling of the brain, which may
result in conal herniation.

• Nephritis of varying degree, especially in Angora goats.

• Congestion and/or oedema of the abomasal folds are a regular finding in cattle, but less so
in sheep and goats.

DIAGNOSIS

Identification of the agent

• Heartwater is often diagnosed by observing E. ruminantium colonies in the brain or intima of blood
vessels. Brain smears are air dried, fixed with methanol and stained with Giemsa. E. ruminantium can
also be detected in formalin-fixed brain sections using immunoperoxidase techniques.

• Polymerase chain reaction (PCR) techniques are available to reveal the presence of E. ruminantium
in the blood of animals with clinical signs, in the tick vector, and to a lesser extent in the blood or bone
marrow of carrier animals. In cultures, the organism is identified by microscopic examination, or by
immunofluorescence/immunoperoxidase staining. In some cases, heartwater may be diagnosed by
inoculating fresh blood into a susceptible sheep or goat.

Serological tests

• Serological tests available include indirect fluorescent antibody tests, enzyme linked immunosorbent
assays (ELISAs) and Western blot. However, when the whole E. ruminantium is used as antigen,
cross-reactions with Ehrlichia spp. occur in all of these tests. Serology has limited diagnostic
applications.

PREVENTION AND CONTROL

• As E. ruminantium cannot survive outside a living host for more than a few hours at room
temperature, heartwater is usually introduced into free areas by infected animals, including
subclinical carriers, or by ticks.

• In heartwater-free countries, susceptible ruminants from endemic regions are tested before
importation. All animals that may carry Amblyomma, including non-ruminant species, must be
inspected for ticks before entry.

• Ticks may be carried into a country on illegally imported animals or migrating birds.

• Outbreaks are usually controlled with quarantines, euthanasia of infected animals and tick control.

• During an outbreak, ticks should not be allowed to feed on infected animals.

• Iatrogenic transfer of blood between animals must also be avoided.

• In endemic regions, heartwater can be prevented by tick control and vaccination.

• Animals moved into endemic areas may be protected by prophylactic treatment with tetracycline.
 172

• Vaccination currently consists of infection with a live E. ruminantium strain, then treatment with
antibiotics when a fever develops. Alternatively, the vaccine may be given to young kids or lambs
during their first week of life, or to calves less than 5 to 8 weeks of age.

• Intensive tick control may increase the susceptibility of animals to heartwater, because it eliminates
the immune boosting effect of persistent exposure to small doses of organisms.

• In endemic areas, animals with heartwater can be treated with antibiotics.

• Tetracycline (oxytetracycline at 10 mg/kg or doxycycline at 2 mg/kg) is effective during the early,

febrile stages of this disease, but animals often die before treatment can be administered.
Antibiotic treatment alone is not always successful in later stages.

• Vector control measures aimed at eradication of Amblyomma ticks by acaricide treatment of cattle
and small ruminants

Inactivated vaccines

• Inactivated vaccines using the Gardel strain and subsequently other strains have been produced in
bioreactors for an industrial production following the development of the whole production
process. The Mbizi strain inactivated vaccine is being developed commercially by Onderstepoort
Biological Products in South Africa.

Live attenuated vaccines

• Isolates of attenuated virulence that do not necessitate treatment of animals would be ideal but a
limited number of such attenuated isolates are available. • An attenuated Senegal isolate has been
obtained and shown to confer 100% protection against an homologous lethal challenge, but very
poor protection against a heterologous challenge.

Recombinant vaccines

• Several reports show partial protection of mice using map1 DNA vaccination and an improvement of
protection by vaccination following a prime (plasmid) – boost (recombinant MAP1) protocol.
However, protection of ruminants has never been demonstrated using this strategy.

• In opposition, significant protection of sheep was reported against homologous and heterologous
experimental challenge following plasmid vaccination using a cocktail of four ORFs (open reading
frames) from the 1H12 locus in the E. ruminantium genome. No further results have been
described since then.

• Recombinant vaccines will probably not be available in the near future.

EHRLICHIOSIS IN DOGS

Signs, Diagnosis and Treatment of Ehrlichiosis in Dogs

Ehrlichia is a type of bacteria that infect dogs and other species worldwide, causing a disease
called ehrlichiosis. Ehrlichiosis has also been called tropical canine pancytopenia (and several other
names). Ehrlichia is commonly transmitted by ticks.

Cause
 173

Ehrlichia bacteria infect white blood cells. There are many species of Ehrlichia, which infect a
wide variety of animals, but there are only a few species that affect dogs. A closely related infection
affecting platelets is caused by a bacteria called Anaplasma platys and is sometimes referred to as
ehrlichiosis as well (Anaplasma platys used to be called Ehrlichia platys until recently). Most Ehrlichia
infections are acquired through tick bites. Infection is also possible via blood transfusions.

Risk Factors

Ehrlichiosis occurs worldwide in areas where the ticks that carry the disease are common.
While any dog can be infected, some breeds, most notably German shepherds, are prone to more
serious chronic infections. Retired racing greyhounds from areas where ehrlichiosis is common may
suffer from chronic, undetected infections and should be checked for ehrlichiosis and other tick-borne
diseases when adopted.

Signs and Symptoms of Ehrlichiosis

The symptoms and severity of illness seen with ehrlichiosis depends on the species of
Ehrlichia involved and the immune response of the dog. Generally, Erlichia canis appears to produce
the most severe illness, and infections tend to progress through various stages.

The acute phase occurs within the first few weeks of being infected and is rarely fatal.
Recovery can occur, or the dog can enter a "subclinical phase" which can last for years, where there
are no symptoms. Some dogs, but not all, eventually progress to the chronic phase, where very severe
illness can develop. However, in practice is is difficult to distinguish these phases.

Signs and symptoms of ehrlichiosis may include:

 Fever

 lethargy

 loss of appetite

 weight loss

 abnormal bleeding (e.g., nosebleeds, bleeding under skin - looks like little spots or
patches of bruising) enlarged lymph nodes

 enlarged spleen

 pain and stiffness (due to arthritis and muscle pain)

 coughing discharge from the eyes and/or nose

 vomiting and diarrhea

 inflammation of the eye

 Neurological symptoms (e.g., incoordination, depression, paralysis, etc.)

 Signs of other organ involvement can appear in the chronic form, especially kidney
disease.

Note: Anaplasma platys causes recurrent low platelet counts but tends to produce only mild
symptoms, if any.
 174

Diagnosis of Ehrlichiosis

It can be difficult to confirm a diagnosis of ehrlichiosis. Blood tests typically show a

decreased number of platelets ("thrombocytopenia") and sometimes decreased numbers of red blood
cells (anemia) and/or white blood cells.

Changes in the protein levels in the blood may also occur. Blood smears can be examined for
the presence of the Ehrlichia organisms. If they are present, the diagnosis can be confirmed, but they
may not always show up on a smear.

Blood can also be tested for antibodies to Ehrlichia, though this can sometimes produce
incorrect results. Specialized testing can check for genetic material from Ehrlichia, and while this is
the most sensitive test, it is not widely available and has some limitations as well. Generally, a
combination of lab tests along with clinical signs and history are used to make a diagnosis.

The diagnosis is further complicated by the fact that dogs infected with Ehrlichia may also be
infected with other diseases carried by ticks, such as Babesia, Lyme disease, or Rocky Mountain
Spotted Fever. Infection with a bacteria called Bartonella has also been found in conjunction with
Erlichiosis and other tick borne diseases.The presence of these other diseases can make symptoms
more severe and and the diagnosis more complicated.

Treatment of Ehrlichiosis

Ehrlichiosis responds well to treatment with the anitbiotic doxycycline. Improvement in

symptoms is usually very quick, but several weeks of treatment is usually needed to ensure a full
recovery. In severe cases where blood cell counts are very low, blood transfusions may be needed.
Reinfection is possible as immunity to Ehrlichia bacteria is not long lasting.

Prevention of Ehrlichiosis

Preventing exposure to the ticks that carry Ehrlichia is the best means of preventing
ehrlichiosis. Check your dog daily for ticks and remove them as soon as possible (it is believed that
ticks must feed for at least 24-48 hours to spread Ehrlichia).

This is especially important in peak tick season or if your dog spends time in the woods or tall
grass (consider avoiding these areas in tick season). Products that prevent ticks such as monthly
parasite preventatives (e.g., Frontline®, Revolution®) or tick collars (e.g., Preventic®) can be used; be
sure to follow your veterinarian's advice when using these products. Keep grass and brush trimmed in
your yard, and in areas where ticks are a serious problem, you may also consider treating the yard and
kennel area for ticks.
 175

ANAPLASMA

Bacteria from the genus Anaplasma belong to the Procaryota kingdom, the family
Anaplasmataceae. Anaplasma belong to obligate intracellular mi¬croorganisms, Gram-negative
bacteria, living in the blood cells of mammals. Vertebrates can be their reservoir, i.e. an environment
where the pathogen can live and proliferate for many years. However, in many cases bacteria from the
genus Anaplasma cause diseases in domestic animals and people.

Taxonomical position of bacteria from the genus Anaplasma

The genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia include obligate intracellular
bac¬teria, parasitizing in the vacuoles of cells in eu¬karytic hosts. Animal pathogens were attributed
to the genus Anaplasma, such as A. centrale, A. marginale, A. platys, A. ovis and A. bovis, and also the
aetio¬logical factor of human anaplasmosis, A. phagocy¬tophilum.

Anaplasma marginale

A. marginale are obligate intracellular bacteria parasitizing in erythrocytes of higher

vertebrates, mostly ruminants.

Anaplasma centrale

A. centrale is a parasite in the erythrocytes of ruminants, mainly cattle. As opposed to A.

marginale, it creates concentrations in the central part of the cell. A. cent¬rale is less pathogenic to
cattle than A. marginale but, most importantly, occasionally gives resistance against the latter. Hence
it is used for the preparation of live vaccine strains, assuring immunological pro¬tection against
bovine anaplasmosis.

Anaplasma bovis

A. bovis is a bacterium detected mainly in cattle, but also observed in small mammals which
are prob¬ably a reservoir of this bacterium. It occurs in monocytes, and the disease it causes is called
monocytic anaplasmo¬sis. The symptoms of the disease are most visible in calves, but also in adult
animals; symptoms include weakening of the body, marked reduction in weight, elevated temperature,
enlargement of prescapular lymph nodes, paling of the mucous membranes, and in many cases an
elevated amount of secreted mucus.

Anaplasma ovis

A. ovis mainly parasitizes small ruminants, i.e. sheep and goats, and its presence has been
confirmed in most regions of the world both in farm and wild animals. Similarly to A. marginale and A.
centrale, these bacteria live in erythrocytes, hence the anaemia of the infected animals.

In the case of A. ovis, bacterial inclu¬sions are found 35–40% of the time in the central or
submarginal part of the erythrocyte of the host and the remaining 60–65% of the time in the marginal
part.
 176

Anaplasma platys

A. platys is mainly a pathogen of canines, usu¬ally dogs. It lives in blood cells, causing Canine
Cyclic Thrombocytopenia (CCT). Clinical symptoms of this disease vary depending on the species of
the dog, but fundamentally CCT causes weakening of the animal, lethargy, anorexia, res¬piratory
distress, fever, increased mucous secre¬tion, purulent ocular discharge, splenomegaly and muzzle
hyperkeratosis. These bacteria do not cause death, and treatment with suitable antibiotics is usually
successful after four weeks. Doxycycline is one of such effective antibi¬otic.

Anaplasma phagocytophilum

A. phagocytophilum is a frequently described bacterium from the genus Anaplasma. It has

been detected in domestic animals, mostly small rumi¬nants, but also in people. These bacteria are
an aetiological factor of granulocytic anaplas¬mosis which in the case of human infections is
de¬fined as Human Granulocytic Anaplasmosis – HGA (Human Anaplasmosis – HA).

In the case of farm animals anaplasmosis caused by A. phagocytophilum is a disease with

subclinical or heavy symptoms. It has unspecific symptoms, such as fever, drowsiness, anorexia,
abortions, drop in body weight and reduction in milk production. Left unattended, especially in weaker
individuals, it can lead to death.

ANAPLASMOSIS IN BEEF CATTLE

Anaplasmosis is an infectious disease of cattle caused by several species of the blood

parasite Anaplasma. A. marginale is the most common pathogen of cattle. Sheep and goats are much
less commonly affected. Anaplasmosis is also called “yellow bag” or “yellow fever” as affected
animals can develop a jaun¬diced appearance.

Transmission

A. marginale can be transmitted two different ways. First, it can be transmitted mechanically
when red blood cells infected with A. marginale are inoculated into susceptible cattle. This can occur
through needles, dehorners, ear taggers, castrating knives or other sur¬gical instruments, and tattoo
instruments.

Mechanical transmission can also occur through the mouthparts of biting insects, such as
biting flies. Face flies, houseflies, and other non-biting insects do not transmit the disease. Horn flies,
although they bite, typically do not go from animal to animal so they are not thought to spread
Ana¬plasma. Mechanical transmission of infected red blood cells must occur within a few minutes of
the blood leav¬ing the infected animal, as the blood parasite does not survive more than a few
minutes outside the animal.

Once susceptible cattle are infected with Anaplasma, the organism multiplies in the
bloodstream and attaches to the animal’s red blood cells. The animal’s immune system destroys the
infected red blood cells in an attempt to fight off the infection. Unfortunately, unin¬fected blood cells
are also destroyed. When the num¬ber of blood cells being destroyed exceeds the number of blood
cells that the body can produce, the animal becomes anaemic. It takes 3 to 6 weeks for clinical signs
to appear after the animal is infected.

Outbreaks

Although many outbreaks of anaplasmosis occur in the spring and summer, they can occur at
any time of the year. The many ways it can be transmitted and the potential for carrier animals makes
the source of an outbreak confusing.
 177

Pathogenesis

Stages of the Disease

Anaplasmosis can be divided into four stages: incubation, developmental, convalescent, and
carrier. These stages and the symptoms associated with them are described below.

Incubation Stage

The incubation stage begins with the original infection with A. marginale and lasts until 1
percent of the animal’s red blood cells (RBCs) are infected. The average incubation stage ranges from
3 to 8 weeks, but wide variations have been documented.

Most variation is directly related to the number of organisms introduced into the animal. After
gaining entry into a susceptible animal, the anaplasma parasite slowly reproduces in the animal’s
blood during the incubation phase. During this period the animal remains healthy and shows no signs
of being infected. Finally, after the parasite has reproduced many times and established itself in the
RBCs of the animal, the body attempts to destroy the parasite.

Developmental Stage

During the developmental stage, which normally lasts from 4 to 9 days, most of the
characteristic signs of anaplasmosis appear. Clinical signs begin to be expressed about halfway
through this phase. As the infected animal’s body destroys the parasite, RBCs are destroyed as well.
When a substantial loss of RBCs has occurred, the animal will show signs of clinical anemia. The
o o o o
body temperature will commonly rise to 104 to 107 F (40 to 41 C), and a rapid decrease in milk
production will occur in lactating cows.

Cattle producers first notice the anemic, anaplasmosis-infected animal when it becomes
weak and lags behind the herd. It refuses to eat or drink water. The skin becomes pale around the
eyes and on the muzzle, lips, and teats. Later, the animal may show constipation, excitement, rapid
weight loss, and yellow tinged skin. The animal may fall or lie down and be unable to rise. Affected
cattle either die or begin a recovery 1 to 4 days after the first signs of the disease.

As a general rule, unless infected cattle can be detected during the early developmental stage,
they should not be treated. There are two primary reasons for this practice. First, if the animal is
forced to move or becomes excited, it may die of anoxia (lack of oxygen in the animal’s system).
Second, antibiotic treatments do little or nothing to affect the outcome of the disease when given
during the late developmental or convalescent stage.

Convalescent Stage

Cattle that survive the clinical disease lose weight, abort calves, and recover slowly over a 2-
or 3-month period. This is known as the convalescent stage, which lasts until normal blood values
return. This stage is differentiated from the developmental stage by an increase in the production of
RBCs (erythropoiesis) in the peripheral blood, shown in an increase in hemoglobin levels and high
total white blood cell counts, among other characteristics. Death losses normally occur during the late
developmental stage or early convalescent stage.

Cattle of all ages may become infected with anaplasmosis, but the severity of illness
increases with age. Calves under 6 months of age seldom show enough signs to indicate that they
are infected. Cattle 6 months to 3 years of age become increasingly ill, and more deaths occur with
advancing age. After 3 years of age, 30 to 50 percent of cattle with clinical anaplasmosis die if
untreated.

Carrier Stage
 178

Unless adequately medicated, cattle that recover from anaplasmosis remain reservoirs
(carriers) of the disease for the rest of their lives. During the carrier stage, an animal will not exhibit
any clinical signs associated with the persistent low-level A. marginale infection.

Nevertheless, the blood from these recovered animals will cause anaplasmosis if introduced
into susceptible cattle. Carriers very rarely become ill with anaplasmosis a second time. Unidentified
carriers in a herd are the most likely source of infection for future outbreaks of the disease.

Clinical Signs

Anaplasmosis is unusual because the clinical signs are most severe in adult animals. Calves
less than a year old that are infected with A. marginale usually do not show clinical signs of the
disease, but will become car¬riers. Carrier animals have immunity against anaplas¬mosis, so even if
they are infected later in life, they will generally not get sick. Cattle 1 to 3 years old will show
increasingly more severe clinical signs. Recovered ani¬mals will also become carriers. Newly infected
adult cattle over 3 years will show the most severe clinical signs, and 30 percent to 50 percent will die
if they are not treated early.

Unless cattle are being watched carefully, dead cows are frequently the first thing noticed
with an anaplas¬mosis outbreak. If cattle are carefully observed, weak¬ness may be the first clinical
sign that is noticed with anaplasmosis. Infected cattle will fall behind the rest of the herd and will not
eat or drink. Cows with light skin will initially look pale around the eyes and muzzle, but later this can
change to a yellowish colour (jaundice). This jaundice is due to the destruction of the blood cells and
their contents being released into the blood stream. Weight loss is rapid. Cattle can become
extremely aggressive if they are oxygen deprived due to the severe anaemia. Oxygen deprivation can
also result in abor¬tions in pregnant cows. Constipation, high fever, and laboured breathing can also
be seen. The most critical period is the first 4 to 9 days after clinical signs appear. Cows that survive
this period have an increased chance of survival.

Diagnostic Tests

Three tests are routinely used to detect the presence of anaplasma antibodies in blood. They
are the complement fixation (CF), fluorescent antibody (FA), and the rapid card agglutination (RCA)
tests.

Treatment

Treatment of anaplasmosis is most effective if given in the early stage of the disease. A
single dose of long-acting oxytetracycline (ex. LA-200®) is administered subcutaneously at 9 mg per
pound of body weight. Blood transfusions are occasionally used. Animals in later stages of the
disease may be so anemic that the stress of handling them will kill them. There is also evidence that
antibiotics at this stage are not effective. Therefore, for very weakened or bellig¬erent cattle,
antibiotic treatment is not recommended.

All affected animals should be provided with easy access to food and water and a low-stress
environment. It may take surviving animals up to 3 months to com¬pletely recover from the disease.
Animals treated with a single dose of antibiotics and those not treated at all will both become carrier
animals. Carrier animals can be cleared of anaplasmosis with repeated injections of long-acting
oxytetracycline or prolonged feeding of chlortetracycline.

Possible antibiotic treatment protocols for eliminating the carrier state are described below:

 Oxytetracycline (50 to 100 mg/ml): 5 or 10 day treatment. 10 mg per pound of body weight
(BW) daily for 5 days or 5 mg per pound of BW for 10 days.
 179

 Intramuscular: To ensure adequate absorption of the medication and prevent excessive

muscle inflammation, do not inject more than 10 ml per injection site.

 Intravenous: Oxytetracycline should be diluted with physiological saline or administered by a

veterinarian.

Oxytetracycline (LA-200): 4 treatments at 3-day intervals.

Each animal receives 4 treatments of LA- 200 at 3-day intervals at a dosage of 9 mg per
pound of BW. The total dose should be divided between two injection sites and given by deep
intramuscular injection.

Chlortetracycline: 60-day treatment.

Chlortetracycline fed at a level of 5 mg per pound of BW daily for 60 days will eliminate the
carrier state of anaplasmosis. Oral administration permits treatment on a herd basis and the use of
economical antibiotic premixes.

Chlortetracycline: 120-day treatment.

Chlortetracycline fed at the rate of 0.5 mg per pound of BW per day for 120 days will eliminate
the carrier state of anaplasmosis. Attempts to eliminate the carrier state of anaplasmosis by feeding
chlortetracycline at the rate of 1 mg per pound of BW every other day for 60 feedings (120 days) did
not consistently rid animals of the A. marginale infections.

General Control Programs

Vaccinating will not prevent susceptible cattle from becoming infected either, but will reduce
the clinical signs of the disease. Vaccination requires a first injection and a booster 4 weeks later.
Both injec¬tions must be completed 2 weeks before the vector season, and the manufacturer
recommends an annual booster vaccine.

RICKETTSIALES

Organisms in the order Rickettsiales form a diverse group of small (0.3 to 0.5 x 0.8 to 2.0 pm),
non-motile, pleomorphic Gram-negative bacteria which replicate only in host cells. They can be
cultured in the yolk sac of embryonated eggs or in selected tissue culture cell lines. Because they
stain poorly with aniline dyes, these organisms should be stained by Romanowsky methods such as
Giemsa or Leishman. In addition to host-cell dependence and poor affinity for basic dyes, a
requirement for an invertebrate vector distinguishes them from conventional bacteria and the
Chlamydiales.

EPIDEMIOLOGY

Animal hosts and arthropod vectors are the reservoirs for most rickettsiae. A number of
rickettsia1 organisms, including Ehrlichia canis, Anaplasma marginale and Haemobartonella felis
produce latent infections. In arthropods, rickettsiae replicate in the epithelial cells of the gut before
spreading to other organs, including the salivary glands and ovaries where further replication may
occur.

Organisms are transmitted when the arthropod feeds on the animal host. Some organisms
such as Rickettsia rickettsii are maintained in a tick population by transovarial transmission. Trans-
stadial but not transovarial transmission of E. canis and E. phagocytophila occurs in ticks.
 180

With the exception of Coxiella burnetii, which produces endospore-like forms and can remain
viable in dust for up to 50 days, most rickettsiae are labile outside host cells. Aerosol transmission of
C.burnetii commonly occurs in domestic animals and humans. In addition, a silent cycle involving
ticks and small wild mammals may constitute a possible source of infection for some domestic
species.

PATHOGENESIS AND PATHOGENICITY

Many Rickettsia species including the causal agents of typhus (R. prowuzekii), murine typhus
(R. typhi) and scrub typhus (R. tsutsugamushi) are primarily human pathogens. Rocky Mountain
spotted fever, caused by Rickettsia rickettsii, which is a common rickettsia1 disease of humans, also
affects dogs. These highly pathogenic organisms have a predilection for the endothelial cells of small
blood vessels. Rickettsia species produce phospholipase which damages the membranes of
phagosomes allowing the organisms to escape into the cytoplasm. Replication in the cytoplasm
induces cytotoxic effects.

Ehrlichia species, with the exception of the human pathogens E. chaffeensis and E. sennetsu,
are pathogens of domestic and feral animals. They have a predilection for either leukocytes or
platelets and they survive and replicate in phagosomes by inhibiting phagosomellysosome fusion.

Cowdria ruminantium, the cause of heartwater in ruminants, probably parasitizes

macrophages and other cell types in lymphoid tissues during the initial phase of infection. Organisms
finally localize in membrane-bound vacuoles in endothelial cells throughout the body.

Two species in the genus Neorickettsia cause acute febrile disease in dogs. These
organisms, which localize predominantly in lymph nodes, produce a generalized lymphadenopathy.
Coxiella burnetii grows preferentially in the acid environment of phagolysosomes and many of its
metabolic activities are detectable only at pH 5 or lower. This pathogen localizes and replicates in
cells of the female reproductive tract and mammary glands of ruminants.

Members of the Anaplasmataceae have a predilection for erythrocytes. Anaplasma species

and Aegyptianella pullorum are found within vacuoles in red blood cells, whereas Haemobartonella
and Eperythrozoon species are located on erythrocyte surfaces. Although H. felis does not usually
penetrate red cells, it may erode surface membranes, increasing osmotic fragility of the erythrocytes
and shortening their life span. Attachment of the organism to the surface of red cells appears to alter
their surface antigens, stimulating autoantibody production and immune-mediated injury to the red
cells. Anaemia, due to H. felis infection results from a combination of haemolysis and premature
removal of red cells from the circulation.

CLINICAL INFECTIONS

Rickettsia1 organisms are relatively host-specific. Because definitive arthropod or fluke

vectors are involved in the transmission of most rickettsiae, diseases associated with these
organisms tend to occur in defined geographical regions. In many instances the clinical signs reflect
the targeting of a particular cell type by the causal agent of rickettsia1 disease. Q fever and Rocky
Mountain spotted fever are important zoonotic diseases.

ROCKY MOUNTAIN SPOTTED FEVER IN DOGS

Rocky Mountain spotted fever, caused by Rickettsia ricketlsii, affects mainly humans and
dogs. In North America, the main tick vectors are Dermacentor variabilis and D. andersoni.
Rhipicephalus sanguineus and Amblyomma cajennense are the main vectors in Central and South
America. Ticks acquire the pathogen while feeding on infected small wild mammals. Rickettsia
rickettsii is maintained in the tick population by transovarial and transstadial transmission. An
infected tick must remain attached for up to 20 hours before salivary transmission to the host occurs.
 181

The organisms, which replicate in endothelial cells of infected dogs, produce vasculitis, increased
vascular permeability and haemorrhage.

Clinical signs

The incubation period of the disease is 2 to 10 days and the course is usually less than 2
weeks. Clinical signs include fever, depression, conjunctivitis, retinal haemorrhages, muscle and joint
pain, coughing, dyspnoea and oedema of the extremities. Neurological disturbance, which occurs in
about 80% of affected dogs, presents as stupor, ataxia, neck rigidity, seizures and coma. Dogs with
mild disease and those treated early in the infection usually recover. In severe disease, death may
result from cardiovascular, neurological or renal damage. At postmortem there is widespread
haemorrhage, splenomegaly and generalized lymphadenopathy.

Diagnosis

Rocky Mountain spotted fever should be considered in dogs with systemic disease which
have been exposed to ticks in endemic areas. Indirect fluorescent antibody test or ELISA
demonstrating a rising antibody titre to R. rickettsii is diagnostic. Antibodies are not demonstrable
until at least 10 days after infection. A marked thrombocytopenia and leukopenia may be present
during the acute phase of the disease. The disease must be differentiated from acute canine
monocytic ehrlichiosis.

Treatment and control

Tetracycline therapy, which usually produces clinical improvement within 24 hours, must be
continued for 2 weeks. Supportive therapy is necessary for severely debilitated dogs. Frequent
removal of ticks is recommended. Because the disease is zoonotic, gloves should be worn during this
procedure.

CANINE MONOCYTIC EHRLICHIOSIS

Canine monocytic ehrlichiosis, a generalized disease of Canidae caused by Ehrlichia canis, is

confined to tropical and subtropical regions. Rhipicephalus sanguineus, the brown tick, is one of the
main vectors and trans-stadia1 transmission occurs. After detachment from an infected host, ticks
can transmit the agent to susceptible dogs for up to 5 months. Dogs often remain carriers for more
than 2 years after recovery from acute disease. Human ehrlichiosis is caused by E. chaffeensis which
is closely related to E. canis.

Clinical signs

Following an incubation period lasting up to 3 weeks, the disease can progress through acute,
subclinical and chronic phases. The acute phase, in which signs range from mild to severe, is
characterized by fever, thrombocytopenia, leukopenia and anaemia. Most affected dogs recover but
some progress to a subclinical phase lasting months or years during which low blood cell values
persist but clinical signs are minimal.

A minority of these dogs later develop a severe form of the disease known as tropical canine
pancytopenia. Persistent bone marrow depression, along with haemorrhages, neurological distur-
bance, peripheral oedema and emaciation are characteristic of this phase of the disease. Hypotensive
shock may ultimately develop, leading to death. Progression to this chronic phase of the disease may
be influenced by factors such as breed susceptibility, immunosuppression and the virulence of the
infecting strain of E. canis.

Diagnosis
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Typical clinical and haematological features in dogs exposed to ticks in an endemic area may
suggest canine monocytic ehrlichiosis. Morulae of E. canis may be detected in mononuclear cells in
Giemsa-stained smears of the buffy-mat layer prepared from peripheral blood.

Seroconversion can be demonstrated 3 weeks after infection using indirect

immunofluorescence. Antibody titres of 1:10 or greater are considered to be indicative of infection.
Ehrlichia canis can be cultured in a canine macrophage cell line.

Treatment and control

Doxycycline therapy for 10 days is recommended. Tetracyclines and chlordmphenicol are

also effective. Fluid replacement therapy or blood transfusions may be necessary. Tetracyclines can
be administered to susceptible dogs entering an endemic area as a short-term prophylactic measure.

CANINE GRANULOCYTIC EHRLICHIOSIS

This disease, recently described in the USA, is caused by Ehrlichia ewingii. Nentrophils are the
primary target cells for the pathogen. Infected dogs, which exhibit mild clinical signs, recover
uneventfully.

CANINE CYCLIC THROMBOCYTOPENIA

'Ehrlichia platys', the cause of this condition, parasitizes platelets. Infected dogs, with
thrombocytopenia recurring at intervals of about 10 days, are usually asymptomatic. Seromnversion,
detected by indirect immunofluorescence, can be demonstrated about 2 weeks after infection.

POTOMAC HORSE FEVER

Potomac horse fever, also known as equine monocytic ehrlichiosis and equine ehrlichial
colitis, is caused by Ehrlichia risticii. Originally described in 1970 in horses near the Potomac River in
Virginia and Maryland, the disease has now been reported throughout North America and in some
European countries. Potomac horse fever occurs during summer months and a fluke vector has been
suggested. Ehrlichia risticii infects epithelial cells of the crypts in the colon and also targets
monocytes, tissue macrophages and mast cells.

Clinical signs

Fever, anorexia, depression, diarrhoea, colic, leukopenia and laminitis may be evident. The
case fatality rate can reach 30%. Transplacental transmission of E. risticii may occur and the agent
may induce abortion. Patchy hyperaemia of the large intestine may be found at postmortem.

Diagnosis

 Clinical signs, although non-specific, may suggest the disease in endemic areas.

 A rising antibody titre detected by indirect immunofluorescence or ELISA tests is consistent

with active infection.

Treatment and control

Oxytetracycline intravenously for 7 days is therapeutically effective. Inactivated vaccines are

commercially available in Northern America.

EQUINE GRANULOCYTIC EHRLICHIOSIS

This disease, often known as equine ehrlichiosis, is caused by Ehrlichia equi. It has been
reported from the USA, some European countries and Israel. Clinical signs include fever, depression,
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ataxia, limb oedema, icterus and petechial haemorrhages on mucous membranes. The disease is
relatively mild, the mortality rate is low and cases tend to occur in late autumn and in winter. The
mode of transmission is not known. Diagnosis is based on the demonstration of morulae of E. equi in
neutrophils during the acute phase of the disease. Elevated antibody titres, demonstrated by indirect
immunofluorescence, and marked leukopenia are additional indicators of infection. Tetracycline
therapy is effective.

BOVINE PETECHIAL FEVER

Bovine petechial fever, also called Ondiri disease, which occurs in both wild and domestic
ruminants is caused by 'Ehrlichia ondiri'. Clinical disease is most common in cattle imported into
endemic areas. The disease is limited to highland areas of Kenya and other East African countries,
and the vector is considered to be a species of tick with restricted distribution. 'Ehrlichia ondiri' is
thought to replicate initially in the spleen and to spread subsequently to other organs.

Clinical signs include high fluctuating fever, depressed milk yield and widespread petechiation
of visible mucous membranes. Oedema and petechiation of the conjunctiva produces 'poached-egg'
eye, a feature typical of severe cases. Death often results from pulmonary oedema. Recovered
animals which become carriers are resistant to reinfection for at least 2 years. The organisms are
often found in granulocytes in smears of peripheral blood stained by the Giemsa method.
Tetracyclines are effective only when administered during the incubation period of the disease.

TICK-BORNE FEVER

Tick-borne fever is a rickettsia1 disease of domestic and wild ruminants caused by Ehrlichia
phagocytophila. The disease tends to be endemic on certain tick-infected upland farms in some
European countries. The main vector is the tick, Ixodes ricinus, in which trans-stadial transmission
occurs. Transmission to ruminant hosts occurs through the bites of infected ticks and, less
commonly, through contaminated instruments. Recovered animals remain infected for up to 2 years
and act as reservoirs of infection for ticks. These carrier animals are immune to challenge with the
homologous strain of E. phagocytophila. Since the maintenance of immunity appears to be related to
repeated exposure to E. phagocytophila, removal of animals from tick-infected pastures results in a
decline in protective immunity.

Clinical signs

Clinical signs, which develop after an incubation period of up to 13 days, include fever,
inappetence and a reduced growth rate in young animals. A drop in milk production and abortions or
stillbirths may occur in naive, pregnant animals after transfer to farms where the disease is endemic.
Most affected animals recover within hvo weeks. However, E. phagocytophila depresses both
antibody-mediated and cell-mediated immune responses, increasing the susceptibility of young lambs
to tick pyaemia and louping ill, diseases which are also tick-transmitted. Haematological changes in
tickborne fever include leukopenia and transient thrombocytopenia.

Diagnosis

The disease should be considered in sick ruminants on tick-infected pastures in endemic

regions. In Giemsa-stained blood smears, more than 70% of neutrophils contain intracytoplasmic blue
morulae during the febrile period of the disease. Inciirect immunofluorescence is used to detect rising
antibody titres.

Treatment and control

 184

Affected lactating cows should be treated with oxytetracycline. Tick control is an essential
part of disease prevention. Long-acting tetracyclines, administered to lambs in the first 2 to 3 weeks
of life, may protect against infection with E. phagocytophila.

ELOKOMIN FLUKE FEVER

'Neorickettsia elokominica', the cause of Elokomin fluke fever, i s morphologically

indistinguishable from N. helminthoeca and has the same fluke vector. The disease is milder than
salmon poisoning disease and has a wider host range which includes Canidae, bears, racoons and
ferrets. 'Neorickettsia elokominica' infection may be concurrent with N. helminthoeca infection and
there is no cross-protection between the two organisms.

BOVINE ANAPLASMOSIS

Bovine anaplasmosis, or gall sickness, caused by Anaplasma marginale, affects cattle in

tropical and subendemic area may suggest the condition. Giemsa-stained blood smears may contain
denselystained bodies (0.3 to 1.0 pm in diameter) located near the periphery of erythrocytes. The
organisms are most numerous about 10 days after the onset of fever, when up to 50% of erythrocytes
can be affected.

The organisms can be identified in blood smears by immunofluorescence. A radioactive RNA

probe and a polymerase chain reaction-based method are sensitive techniques used for detecting the
pathogen.

Serological tests are of particular value in detecting latent infections. These tests include
complement fixation test, card agglutination test, ELISA and dot enzyme-linked immunosorbent assay.

Treatment and control

Long-acting oxytetracycline or imidocarb dipropionate, administered early in the disease, are

effective. Supportive therapy is essential in severe cases. In endemic areas, control measures are
aimed at minimizing stress in indigenously-reared cattle. Prior to introduction into an endemic region,
animals must be vaccinated. A live A. centrale vaccine, which provides partial protection against A.
marginale, is used only in calves. Attenuated and inactivated A. marginale vaccines are also available.

CHLAMYDIA AND CHLAMYDOPHILA SPECIES

Chlamydiae are obligate intracellular bacteria with an unusual developmental cycle during
which unique infectious forms are produced. They replicate within cytoplasmic vacuoles in host cells.
 185

On account of their apparent inability to generate ATP, with resultant dependence on host cell
metabolism, they have been termed 'energy parasites'.

The family Chlamydiaceae belongs to the order Chlamydiales. Currently two genera,
Chlamydia and Chlamydophila, and nine species are described. Formerly a single genus and four
species, Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, were recognised. This
classification was based on phenotypic characteristics such as host preference, inclusion
morphology, iodine staining for the presence of glycogen, and sulphonamide susceptibility. However,
recent nucleic acid sequencing studies of the 16s and 23s rRNA genes confirm two distinct lineages.

In the developmental cycle of chlamydiae, infectious and reproductive forms are

morphologically distinct. Infectious extracellular forms, called elementary bodies (EBs) are small (200
to 300 nm), metabolically inert and osmotically stable. Each EB is surrounded by a conventional
bacterial cytoplasmic membrane, a periplasmic space and an outer envelope containing
lipopolysaccharide.

The periplasmic space does not contain a detectable peptidoglycan layer and the EB relies on
disulphide cross-linked envelope proteins for osmotic stability. Elementary bodies enter host cells by
receptor-mediated endocytosis. Acidification of the endosome and fusion with lysosomes are
prevented by mechanisms which are not fully understood. A process of structural reorganization
within the pathogen, of several hours duration, results in the conversion of an EB into a reticulate body
(RB). The RB, about 1 µm in diameter, is metabolically active, osmotically fragile and replicates by
binary fission within the endosome. The endosome and its contents, when stained, is called an
inclusion. When a number of inclusions containing RBs of C.trachomatis are formed in an infected
cell, fusion of these structures may occur.

USUAL HABITAT

The gastrointestinal tract appears to be the usual site of Chlamydophila species infection in
animals. Intestinal infections are often subclinical and persistent. Faecal shedding of the organisms,
which is typically prolonged, becomes intermittent with time. The EBs can survive in the environment
for several days.

PATHOGENESIS AND PATHOGENICITY

Chlamydiae infect over 130 species of birds and a large number of mammalian species
including man. In recent years isolations have also been made from invertebrate species. Chlamydia1
species are usually associated with specific diseases in particular hosts. In sheep, C.abortus is an
important cause of abortion whereas infections with C. pecorum are frequently inapparent.
Interspecies transmission is uncommon. When it occurs, the outcome of infection in the secondary
host may be either similar, as in transmission from sheep to cattle, or severe, as in transmission from
sheep to pregnant women. Infection with C.pecorum is associated with conjunctivitis, arthritis and
inapparent intestinal infection. The type of clinical presentation relates to the route of infection and
the degree of exposure. Environmental factors and management practices can influence the
prevalence of some chlamydial infections such as enzootic abortion in ewes, which tend to be more
prevalent in intensively managed lowland flocks.

Many chlamydial infections are asymptomatic, particularly when they are localized in
superficial epithelia. They may also persist for long periods without inducing protective immunity.
However, chronic infections may repeatedly stimulate the host immune system. Chlamydiae possess
a number of heat shock proteins which are partially homologous to heat shock proteins in other
bacteria and to a number of human mitochondrial proteins.

DIAGNOSTIC PROCEDURES
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Specimens for isolation of the organism should be placed in suitable transport medium such
as sucrosephosphate-glutamate medium supplemented with foetal calf serum, aminoglycoside
antibiotics and an antifungal agent. As chlamydiae are thermolabile, samples should be kept at 4°C.
For long term storage, samples should be frozen at -70°C. However, each cycle of freezing and
thawing reduces the titre of the stored organisms.

Direct microscopy is suitable for the detection of the organisms in smears or tissue sections
containing moderate numbers of organisms. Placental smears from cases of chlamydial abortion
typically contain large numbers of organisms. Suitable chemical staining procedures include the
modified Ziehl-Neelsen, Giemsa, modified Machiavello and Castaneda methods. Methylene blue-
stained smears can be examined by darkfield microscopy.

Several commercial kit sets, employing ELISA methodology, have been developed for the
detection of C. trachomatis. Many of these kit sets detect chlamydial lipopolysaccharide (LPS) which
is common to all Chlamydia and Chlamydophila species. Consequently, they can be used to detect the
LPS of species in both genera.

Chlamydiae can be isolated either in embryonated eggs, inoculated into the yolk sac, or in a
number of continuous cell lines such as McCoy, L929, baby hamster kidney and Vero. Tissue culture
cells are usually grown in flat-bottomed vials or in bottles containing coverslips for ease of fixing and
subsequent staining.

Polymerase chain reaction techniques have been developed for the detection of chlamydial
DNA in samples. Several serological procedures are available for the detection of antibodies to
chlamydiae including complement fixation, ELISA, indirect immunofluorescence and micro-
immunofluorescence.

CLINICAL INFECTIONS

A wide range of animal species are susceptible to infections with chlamydiae. Both the
severity and the type of disease produced by chlamydiae are highly variable, ranging from clinically
inapparent infections and local infections of epithelial surfaces to severe systemic infections.
Diseases associated with chlamydia1 infections include conjunctivitis, arthritis, abortion, urethritis,
enteritis, pneumonia and encephalomyelitis. Clinical signs and their severity are influenced by factors
related to both host and pathogen, and one type of clinical presentation usually predominates in
outbreaks of disease.

ENZOOTIC ABORTION OF EWES

Enzootic abortion of ewes (EAE) caused by C. abortus (formerly known as ovine strains of C.
psittaci), is primarily a disease of intensively managed flocks. The disease is economically significant
in most sheepproducing countries. Although abortion associated with C. abortus is best documented
in sheep, it has also been reported in other domestic species including cattle, pigs and goats.
Chlamydial infection in cattle and goats often originates from sheep. The source of infection in pigs is
less clearly defined).

Epidemiology

Infection is usually introduced into clean flocks when infected replacement ewes abort. Large
numbers of chlamydiae are shed in placentas and uterine discharges from affected ewes. Organisms
can remain viable in the environment for several days at low temperatures.

Infection occurs by ingestion. The role of infected rams in venereal spread is uncertain. Ewes
infected late in pregnancy do not usually abort but may do so in the next pregnancy. Infection early in
pregnancy can result in abortion during that pregnancy. Ewe lambs may acquire infection during the
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neonatal period and abort during their first pregnancy. As a result, the most dramatic outbreaks of
EAE often occur in the year following the introduction of infection into a flock.

Clinical signs

Enzootic abortion of ewes is characterized by abortion during late pregnancy or by the birth of
premature weak lambs. Aborted lambs are well developed and fresh. Necrosis of cotyledons and
oedema of adjacent intercotyledonary tissue in affected placentas is often present along with a dirty
pink uterine exudate. Aborting ewes rarely show evidence of clinical disease and their subsequent
fertility is usually unimpaired. Although up to 30% of animals in a fully susceptible flock may abort, a
rate of 5 to 10% is more usual in flocks in which the disease is endemic.

Diagnosis

Aborted lambs and evidence of necrotic placentitis are suggestive of EAE. Large numbers of
EBs can be demonstrated in placental smears using suitable staining procedures. Commercial
diagnostic kits are available for the detection of chlamydial antigen in samples. Isolation of
chlamydiae in suitable cell lines or in the yolk sac of embryonated eggs is possible. Polymerase chain
reaction techniques are available and can be carried out using species-specific primers to distinguish
C. abortus and C. pecorum.

A number of different serological tests can be used for the detection of chlamydial antibodies
including the complement fixation test, ELISA and indirect immunofluorescence. The use of
recombinant antigens specific for C.abortus may improve the specificity of serological tests.

Treatment and control

Chlamydiae are susceptible to a number of antibiotics which can be used during an outbreak.
Administration of long-acting oxytetracycline to in-contact pregnant ewes has been shown to increase
the number of live born lambs. However, antibiotic treatment does not eliminate the infection and
treated ewes may shed chlamydiae at parturition.

Transmission of infection in an affected flock can be reduced by isolating all aborted ewes
for 2 to 3 weeks, removing and destroying all placentas, thoroughly cleaning areas where abortions
occurred and administering long-acting oxytetracycline to ewes which have not yet lambed.

A decision should be made either to vaccinate or attempt to eradicate the disease by culling.
A live attenuated vaccine is available which should be administered to ewes prior to breeding. An
inactivated vaccine is also available which can be used in pregnant animals. Chlamydophila abortus
infection is serious and potentially life-threatening for pregnant women who should avoid contact with
ewes during the lambing season.

FELINE CHLAMYDIOSIS

Chlamydaphila felis (formerly known as feline strains of C.psittaci) is associated with

conjunctivitis and less commonly rhinitis. Feline pneumonitis, the original name for feline
chlamydiosis, is now considered a misnomer because of the rarity of lower respiratory tract infection
caused by C.felis in cats.

Epidemiology

Serological surveys have revealed that up to 10% of cats become infected with C. felis.
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Infection is transmitted by direct or indirect contact with conjunctival or nasal secretions. Organisms
may also be shed from the reproductive tract. Infection may be persistent with prolonged shedding of
organisms and clinical relapses. The stress of parturition and lactation may trigger shedding of
organisms by infected queens facilitating transmission to their offspring.

Clinical signs

After an incubation period of about 5 days, unilateral or bilateral conjunctival congestion, clear
ocular discharge and blepharospasm become evident. If secondary infection with organisms such as
Mycoplasma felis and Staphylococcus species occurs, the ocular discharge may become
mucopurulent. Conjunctivitis may be accompanied by sneezing and nasal discharge. The condition
usually resolves without treatment in a few weeks. However, persistent infection with recurring
clinical episodes also occurs.

Diagnosis

Stained conjunctival smears may reveal intracytoplasmic inclusions. The organism may be
isolated in suitable cell lines or in embryonated eggs. Commercial diagnostic ELISA kits for detecting
the genus-specific lipopolysaccharide antigen are available. Polymerase chain reaction protocols
have been developed for samples. The complement fixation test or the indirect immunofluorescence
test can be used to detect chlamydial antibody titres. However, the level of antibody does not
necessarily correlate with active infection.

Treatment and control

Chlamydiae are susceptible to several antibiotics. All in-contact cats should be treated at the
same time. Modified live vaccines are available for parenteral inoculation. Vaccination reduces the
clinical effects of natural infection but does not prevent infection or the shedding of organisms.
Inadvertent intraocular administration of the vaccine can result in conjunctivitis. A small number of
cases of conjunctivitis in humans involving C. felis have been reported.

AVIAN CHLAMYDIOSIS

Infections with C. psittaci in psittacine birds were origin-ally designated psittacosis and
ornithosis was reserved for chlamydia1 infection in other avian species. Avian chlamydiosis is
currently the preferred designation for the condition. The disease has been recorded worldwide.

Epidemiology

A wide range of both wild and domestic avian species are susceptible to infection. Isolates
can be divided into several serovars on the basis of reactivity with monoclonal antibodies. The
organism is present in respiratory discharges and faeces of infected birds. Infection is usually
acquired by inhalation or by ingestion. Subclinical infection is common. Clinically affected and carrier
birds may shed organisms intermittently for prolonged periods. Stress arising from captivity,
transportation, egg-laying, overcrowding and intercurrent infection is important in precipitating
disease outbreaks.

Clinical signs

Avian chlamydiosis is a generalized infection, affecting particularly the digestive and

 189

respiratory tracts. The incubation period is up to 10 days. Clinical signs vary in nature and severity,
depending on the strain of C.psittaci and the species and age of the affected birds. Signs include loss
of condition, nasal and ocular discharges, diarrhoea and respiratory distress. The most frequent post-
mortem findings are hepatosplenomegaly, airsacculitis, peritonitis.

Diagnosis

Organisms may be identified in stained impression smears of affected tissues. Chlamydial

antigen may be detected using immuno histochemistry or ELISA kits. chlamydial DNA may be
demonstrated by the polymerase chain reaction. Isolation of C.psittaci is carried out in cell culture or
embryonated eggs.

Antibodies to C.psittaci may be detected using suitable serological tests including the
complement fixation test and ELISA. However, interpretation of antibody titres can be difficult
particularly when single samples are tested. Paired serum samples or samples from several birds in a
flock are more reliable for diagnosis.

Treatment and control

Tetracyclines are the antibiotics of choice. An extended course of treatment over several
weeks is required. No commercial vaccines are available. Imported birds, particularly psittacine
species, should be held in quarantine and receive tetracycline medicated feeds. Proper husbandry and
suitable transportation minimize the occurrence of clinical disease. Avian chlamydial isolates are
potentially zoonotic. Infection, which commonly follows aerosol inhalation, may be subclinical or
result in systemic disease. Pulmonary involvement is common. Meningitis or meningoencephalitis
may develop in severely affected individuals.
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EMERGING AND RE-EMERGING DISEASES

Emerging infectious diseases

Emerging infectious diseases are those due to newly identified and previously unknown
infections which cause public health problems either locally or internationally.

Re-emerging infectious diseases

Re-emerging infectious diseases are those due to the reappearance and increase of
infections which are known, but had formerly fallen to levels so low that they were no longer
considered a public health problem.

What causes emergence or re-emergence of infectious diseases?

Several factors contribute to the emergence and re-emergence of infectious diseases, but
most can be linked with the increasing number of people living and moving on earth: rapid and intense
international travel; overcrowding in cities with poor sanitation; changes in handling and processing of
large quantities of food; and increased exposure of humans to disease vectors and reservoirs in
nature. Other factors include a deteriorating public health infrastructure which is unable to cope with
population demands, and the emergence of resistance to antibiotics linked to their increased misuse.

AVIAN INFLUENZA

Avian influenza is a zoonotic, globally important disease of birds that can be categorised as
either low pathogenic (LPAI) or highly pathogenic (HPAI) according to the virulence of the virus in
animals. Outbreaks of LPAI are common around the world but LPAI typically causes no clinical signs
or only minor illness in infected birds. LPAI strains are generally less of a threat to human health; in
addition, LPAI can have significant economic repercussions as a result of export restrictions and
culling of birds, particularly in developing countries where the use of vaccines and other veterinary
medicines is difficult due to weak veterinarian services and small farming units. Compared with LPAI,
HPAI is more readily detected in poultry due to very high levels of mortality in infected birds, and can
cause potentially catastrophic economic consequences, from significantly reduced livestock
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populations to lost export markets. Though relatively rare, sporadic human infections of HPAI have
occurred in cases of close contact with infected birds and caused serious illness and even death.

Transmission and spread

Influenza viruses are shed in the oral, respiratory and faecal secretions of infected birds and
spread via direct contact between healthy and infected birds, or indirectly via contact with
contaminated equipment and people. Transmission to humans leading to clinical disease is a rare
event but can take place in cases of close contact with infected birds or in heavily contaminated
environments. The ability of the virus to infect a diversity of wild birds and other vertebrate hosts
(including pigs), its prolonged environmental survival in favourable conditions, its zoonotic potential
and its capacity for mutation via antigenic drift or antigenic shift, as well as the lack of completely
effective vaccines for use in poultry, make avian influenza a particularly dangerous disease for both
animal and human populations.

Control measures

Various controls are already in place for avian influenza across most of the world, including
biosecurity and surveillance, stamping out, and regional vaccination programmes for poultry in high-
risk areas. As there is no treatment for avian influenza once clinical signs appear, targeted stamping
out without vaccination is the chosen control method in most developed countries, where early
detection and compensation programmes are widespread. Vaccination is typically only implemented
in endemic areas as a preventative or during an outbreak as an adjunct control measure when all
other measures are insufficient. Moreover, vaccination has to be accompanied by surveillance and
movement controls to be effective because although vaccines may reduce the susceptibility,
morbidity and mortality of birds subsequently exposed to infection, they do not prevent the shedding
of potentially infectious levels of the virus.

BLUETONGUE

Bluetongue is a non-zoonotic, non-contagious vector-borne disease, caused by a virus of the

Reoviridae family with 25 known serotypes worldwide. It affects all domestic and wild ruminants, with
sheep and cattle experiencing the highest rates of infection. The severity of the disease depends on
the strain and morbidity can be very high in susceptible animals. Bluetongue outbreaks also cause
direct economic losses through disease and mortality, loss of production, loss of milk yield, and
declines in fertility. As bluetongue is not zoonotic, it poses no risks to human health and cannot be
contracted or spread through food.

Transmission and spread

Bluetongue is transmitted by biting midges that are infected with the virus after ingesting
blood from infected animals. The spread of the disease depends mainly on those ecological and
climatic factors that favour biting midge populations; outbreaks are often seasonal, occurring at or
shortly after the season of peak midge activity. As climate change becomes more of a concern in the
future, the risks of bluetongue causing an epidemic will continue to increase. According to a leading
expert on vector-borne diseases, “through changes to climate, one of the most competent vector
species of midge has spread around southern Europe and further north than it had before” while at
the same time midge vectors have become “better at being able to transmit the virus.”

Control measures
 192

Bluetongue control measures include the surveillance of susceptible animals, quarantine,

zoning, insect control and vaccination. Since controlling midge populations is not possible, they are
too numerous vaccination is the most effective practical measure to minimise losses related to the
disease in endemic regions. Vaccines used against bluetongue are both live attenuated and
inactivated. Live attenuated vaccines, until recently the only bluetongue vaccines commercially
available, are relatively inexpensive and can provide long-lasting protection. However, they are not
always sufficiently weakened and they can actually generate the disease they intend to prevent.
Though more expensive, if properly produced and administered, inactivated vaccines can provide
reliable and protective immunity from bluetongue. An efficient treatment method for infected animals
is still lacking.

WEST NILE FEVER

West Nile fever is the disease caused by West Nile virus (WNV), a mosquito borne virus that
primarily affects birds but also horses and humans. Originally confined to tropical areas, the disease
has recently spread to temperate zones largely as a result of climate-related vector expansion and is
now endemic in most regions of the world. Because of its potential for permanent neurological
problems or death in humans, WNV remains a significant public health risk in endemic areas.

Transmission and spread

WNV is transmitted by mosquitoes, while birds are the most commonly infected animal and
serve as the primary reservoir host. The disease is maintained between these two agents in endemic
regions but it can spread to humans and horses under warm environmental conditions. The vast
majority of human cases are caused by mosquito bites, and the virus cannot be transmitted directly
from person to person. Recent climate change developments favour the emergence of WNV
outbreaks by reducing the time that elapses between the mosquito bite and the transmission time,
which is temperature dependent.

Control measures

Mosquito control and surveillance remain the primary control measures for the prevention of
WNV. There are effective, licensed vaccines for use in horses, including inactivated WNV formulations
and ‘chimeric’ recombinant vaccines, which express WNV proteins from a different virus backbone.
Currently no human vaccines are available, although several vaccine candidates are under
development. Though vaccination of horses means that they are protected from the disease, it does
not break the transmission cycle as mosquitoes (and vaccinated animals) continue to be infected and
capable of spreading the disease.

CLASSICAL SWINE FEVER

Classical swine fever (CSF) is a highly contagious disease caused by a species specific virus
that affects swine but is related to viruses affecting cattle and sheep. Highly virulent strains of the
virus result in high levels of mortality among infected pigs. While the virus cannot be transmitted to
humans and therefore poses no risk for human health, it can pose serious economic threats. In the
developed world, the greatest costs result from trade restrictions imposed on both live pigs and pork
products once an outbreak is reported.

Transmission and spread

Transmission of CSF usually takes place through direct contact between infected animals.
The virus can also be spread through the transportation of animals in contaminated vehicles and
through feed since the virus can survive in pork products for months.
 193

Control measures

In areas where outbreaks have not occurred, control measures include early detection and
reporting, movement controls including strict import policies and quarantining of pigs, and effective
hygiene measures, including protecting domestic pigs from contact with wild boars. Stamping out
remains the main control measure used to prevent the further spread of disease during outbreaks.
While effective vaccines are available, vaccination is used only rarely to limit the spread of the disease
after an outbreak has already occurred. In countries which are free of disease, or where eradication is
in progress, vaccination is normally prohibited. There are no effective treatments for CSF in infected
animals.

EQUINE INFLUENZA

Equine influenza is a highly contagious respiratory disease of horses (and other members of
the equidae family including donkeys and zebras) caused by two main subtypes of influenza type A
viruses (H7N7 and H3N8). Equine influenza outbreaks are typically characterised by a rapid spread of
the virus and very high infection rates in unvaccinated horses. However, clinical signs of illness
usually resolve within a few days without complications and fatalities are rare. The virus does not
cause disease in humans, and its economic impact is primarily due to the highly contagious nature of
the virus and the disruption of equestrian activities.

Transmission and spread

The transmission of the disease occurs through direct contact with infected animals or
through the transmission of the virus on clothing or equipment carried by humans that work with
horses. Equine influenza can spread quickly in susceptible populations and cause outbreaks where
conditions are most favourable, for example when horses are kept in close quarters, during
transportation, and where mixing of horses from different locations occurs. The virus is often spread
across borders by the movement of infected horses.

Control measures

Rapid diagnosis, movement restrictions and vaccination are the key control measures.
Vaccines are widely available and routinely used for competition horses in Europe, the Americas, and
Asia. In some countries vaccination is mandatory for horses that are competing in equestrian events
and the International Federation for Equestrian Sports requires vaccination of horses every six
months. Vaccines have been useful in limiting virus replication, reducing or eliminating clinical
disease and reducing the risks of transmission. However, given strain variation, vaccines are not
always successful in preventing infection. Moreover, vaccines are not always easy to obtain in
developing countries.

TRANS-BOUNDARY ANIMAL DISEASE AND THEIR IMPACTS ON INTERNATIONAL TRADE

Trans-boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the
world and transmissible diseases which have the potential for very serious and rapid spread,
irrespective of national borders, which has serious socio-economic or public health consequence and
they are importance in the international trade of animals and animal products. With rapidly increasing
globalization, an associated risk of movement of trans-boundary diseases is emerging. Trans-
boundary animal diseases represent a serious threat.
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They reduce production and productivity, disrupt local and national economies and also
threaten human health (zoonotic). Trans-boundary animal disease is a concern globally; cumulative
effort is needed at international level to minimize the spread of infectious diseases across the
borders.

Considering that livestock rearing constitutes a significant share in the national economy of a
developing country, it is imperative to take up disease control initiatives. Measures are required to
safeguard the livestock industry from epidemics of infectious diseases and to uphold safe
international trade of livestock and their products. In this regard, it is essential to develop scientific
and risk-based standards that facilitate the international trade in animal commodities.

INTRODUCTION

Trans- boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the
world. Moreover, their economic importance is a major constraint in international trade. Their
implication on human Health and National food security cannot be over emphasized. Zoonotic
diseases among TAD’s include diseases like West Nile Virus (WNV), Rift Valley Fever (RVF), Mad Cow
disease (BSE), Bovine Tuberculosis and Highly Pathogenic Avian Influenza (HPAI). Other important
TADs are Foot and Mouth Disease (FMD), Contagious Bovine Pleuropneumonia (CBPP), Lumpy Skin
Disease, African Swine Fever (ASF) and Newcastle Disease (ND). They have the potential for very
rapid spread, irrespective of national borders and these diseases can cause serious socio-economic
and possibly public health consequences.

Trans-boundary animal disease have a multi casual origin; some factors associated with this
process include: Trade and international travel (increased frequency and speed of local and
international travel, fostered by the globalization process promotes the spread of microorganisms on
a global scale), Changes of agricultural practices (animal domestication was one of the main
promoters of microbial evolution by facilitating the availability of new susceptible hosts at high
densities, due to the intensification of livestock systems), Climate change (which causes changes eco
-geographical distribution of vectors), Reduction of habitat and increased contact with wild animals
and Introduction of naïve wild and domestic animals to new geographic areas where the disease is
endemic and immunologically unknown for them (increases zoonotic pool within a geographic region).

Trans-boundary and emerging diseases are becoming ever more important since they can
spread throughout an entire region, impact trading partners and commerce, tourism, consumer
confidence and occur in distinct countries, with devastating economic and livelihood consequences.
With the globalization of trade and the increasing movements of people, these major crises will
continue to menace the global animal and human which is most of the time in developing countries.

Trans-boundary animal disease has a significant impact on national economies due to its
high rates of morbidity and mortality in the susceptible animal populations, costs of control or
eradication programmes and restrictions on international trade. These diseases negatively affect
livestock or poultry production in the country because they impose heavy economic losses in the form
of morbidity and mortality and are thus considered as vital threat to livestock production and
livelihood of poor farmers, especially in those areas of the country where such diseases have
assumed an endemic role. Livestock enterprises and animal production contribute significantly to the
world economy, provide household source of income, food security, source of energy, draft power for
crop cultivation, high quality animal proteins and vitamins (meat, milk), manure, raw materials (hides
and skins) and bride price and generate a livelihood for 1.0 billion poor people in the world.

Transboundary Animal Disease:

Trans-boundary animal disease are transmissible diseases which have the potential for very
serious and rapid spread, irrespective of national borders, which are of serious socio-economic or
public health consequence and which are of major importance in the international trade of animals
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and animal products. Food and Agriculture Organization of the United Nations, defines Trans-
Boundary Animal Diseases (TADs) as, Those diseases with an essential impact on the economy, trade
and/or food security of a group of countries, which can be easily spread to other countries, reaching
epidemic proportions and that require control and eradication cooperation between different nations.

Trans- boundary animal diseases (TADs), includes those that are zoonotic (infective to humans)
and continue to cause widespread negative socio-economic impacts in an increasingly globalized
world with a huge and increasing volume of regional and international trade in livestock and livestock
products and the rapid movement of large numbers of people across continents through air travel.
Several infectious zoonotic diseases have recently emerged, causing devastating economic losses in
the countries affected.

Ways of Transmission of Trans-Boundary Animal Disease:

Many cases of the Trans -Boundary Animal Diseases (TADs) have been reported in various areas
of the world during last decades. Such TADs are easily transmitted from one country to another, due
to the rapid globalization including the increase of international trade in domestic and wild animals
and animal products, to the expansion of human population, global climate changes, changes of
agricultural production systems and to microbiological adaptation. The common ways of introduction
of animal diseases to a new geographical location are through entry of live diseased animals and
contaminated animal products. Other introductions result from the importation of contaminated
biological products such as vaccines or germplasm or via entry of infected people (in case of
zoonotic diseases). Even migration of animals and birds, or natural spreading by insect vectors or
wind currents, could also spread diseases across geographical borders. Traditionally, trade, traffic
and travel have been instruments of disease spread. Now, changing climate across the globe is
adding to the misery.

International trade in live animals and animal products offers opportunities for pathogens and
vectors to be transported across oceans and continents. However, with the exception of a few
documented examples, such as, the multiplicity of routes of introduction, including active and passive
dispersal of vectors, infected human hosts, animal movements and migration, transportation of
goods and biological invasions such as, introduction, initial dispersal, establishment and spread, the
specific contribution of globalization to disease emergence is inherently difficult to quantify.

Control of Trans-Boundary Animal Disease:

Combating diseases is a necessity for farmers. Though a farmer’s decision to control the
diseases or not is a private one, the presence of an infectious disease in a farm poses a threat too
adjacent and even distant farms and can even affect other animal species and develop into an
epidemic. This situation where high stakes are involved demand the intervention and action from
public agencies or governments.

As TADs are a concern globally, cumulative effort is needed at international level to minimize
the spread of infectious diseases across the borders. Reducing man-made disasters that have
adverse implications on climate, global warming and climate change either due to natural or
anthropogenic influences are likely to predispose the animal population to newer infections.
Therefore, collective efforts are needed to minimize adverse climatic changes.

Interrupting the human-livestock, wildlife transmission of infections

Diseases at the wildlife–livestock interface must become the focus for surveillance of emerging
infectious diseases. Breaking the cycle of disease transmission would help control the spread of
infections. Preventing incidence of trans-boundary animal disease can also be practiced by
controlling disease transmitting vectors, minimizing the movement of animals across the borders and
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prompt practice of quarantine protocol. Geographic information system (GIS) and remote sensing
could be utilized as early warning systems and in the surveillance and control of infectious diseases.

Surveillance and control of infectious diseases

 Establishing regional biosecurity arrangement with capacity for early disease warning system
for surveillance, monitoring and diagnosis of emerging disease threats.

 Undertaking animal breeding strategies to create disease resistant gene pools, enhancing
host genetic resistance to disease by selective breeding of resistant animals is a smart
strategy to improve natural immunity of animals to counter invading infections.

 Strengthening government policies to enhance agricultural/animal research and training and

technology development. More funds need to be allocated for this purpose to build goal
oriented research programs in combating TADs.

 Ensuring appropriate preparedness and response capacity to any emerging disease. Keeping
in view that emerging infectious diseases are a constant threat, detection capacity and then
implement a timely response.

Challenges in Dealing with Trans-Boundary Animal Disease:

 Requirement of novel systems having capacity of real-time surveillance of emerging diseases;

for this, need driven research and service oriented scientific technology are a necessary at
regional levels. Research emphasis has to be on specific detection and identification of the
infectious agents.

 Need for epidemiological methods to assess the dynamics of infections in the self and
neighbouring countries/regions. These methods should be of real-time utility. Research and
development of disease diagnostic reagents those do not need refrigeration is also important.

 More importantly, they should be readily available as well as affordable, for use in pen-side
test format.

 There are many diseases for which there is inadequate supply of vaccines or there are no
vaccines available. Insufficient or lack of vaccine hampers the disease control programmes.

 Even if a technology is available, it has to be cheaper to adopt at the point of use. Need for
ensuring public awareness of epidemic animal diseases. Many farmers are unaware of the
emerging diseases.

 As such, unless reported to concerned regional authority, an emerging disease may go

unnoticed. Shortage of government and private funding for research on emerging animal
disease problems, Government as well as industries dealing with animal health should take
initiative and appropriate sponsorship in these regard.

 Inadequate regulatory standards for safe international trade of livestock and livestock
products. Otherwise, there would be a compromised situation in disease control strategies.
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Impact of Transboundary Animal Disease on International Trade:

Trans-boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the
world. Moreover, their economic importance is a major constraint in international trade losses
themselves. Therefore, the countries which are free from major diseases will tend to protect their
local agriculture by totally excluding the importation of livestock products from areas affected by
specific animal diseases. Conversely, benefits of elimination of trans-boundary animal diseases can
be very large as it offers the opportunity of gaining access to high-value export markets. Trans-
boundary animal disease also has implications on domestic trade as the veterinary authority imposes
restriction on animal movement as a part of disease control measure. In evaluating TAD impacts, it is
now recognized that poverty implications, technical feasibility and political desirability play a role in
disease response program selections in LDCs Furthermore, the cost benefit offset of eradication
versus management programs will vary by disease, where high impact diseases do not necessarily
carry high benefits from eradication.

Trans-boundary animal diseases including those that are zoonotic (infective to humans),
continue to cause widespread negative socio-economic impacts in an increasingly globalized world.
With a huge and increasing volume of regional and international trade in livestock and livestock
products and the rapid movement of large numbers of people across continents through air travel.
Several infectious zoonotic diseases have recently emerged, causing devastating economic losses in
the countries affected. These have a wide-ranging impact on the livelihoods of farmers and on
regional and international trade, food safety, public health and international travel and tourism.
Disease pathogens continue to evolve and adapt themselves to animals and humans alike. Disease
investigation indicates that many of these new diseases emerge in response to number critical
factors, such as changes in climate, ecosystems, animal production systems and land use, all of
which alter the interactions between pathogens and various hosts.

Loss of production and productivity is likely to influence the market price of the commodity,
determined by the supply and demand effects induced by TADs. Limited supply can result in increase
in market price but on the contrary public health concern associated with certain TADs may also
decrease the demand. Moreover, relative effects on producers and consumers of the production
shortfall will depend on the relative elasticities of demand and supply. Another dimension of the price
and market effects is that it is not only the producers but also the traders who are directly affected
international trade in livestock and livestock products continues to be seriously hindered by epizootic
animal diseases, in particular those categorized as‘transboundary animal diseases’ (TADs). There is
the danger that informal and illegal trans-border trade in livestock which abound in some countries
could derail the revolution.

Economic Impacts:

Trans-boundary animal diseases (TADs) cause significant economic losses throughout the
world. But producers in less developed countries (LDCs) are at particular risk because livestock
provide not only income and an asset base, but also food, draught power and various social functions.
Trans-boundary diseases threaten food security, affect livelihoods of rural communities and disrupt
local and international trade.

In general, livestock´s products have increased their international trade in 4% only in the last two
decades. However, as a result of the emergence and re-emergence of various animal diseases, such
as bovine spongiform encephalopathy (BSE), the annual growth of meat products decreased 2% in the
late 1990s. Therefore, the cost of trans-boundary animal diseases relates to agricultural products, to
the country’s economy and international markets are massive. Thus, it is very important to create
public policies focused to assure countries’ food security (especially in developing nations) to avoid
negative economic impacts caused by TADs, especially on the more susceptible social stratus. The
World Bank has estimated that zoonotic disease outbreaks in the past 10 years have cost worldwide
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more than $US200 billion due to loss of trade, tourism and tax revenues.

Pests and animal diseases cause the loss of more than 40% in the global food supply, being a
clear threat to the residual economies of developing countries and food security of its inhabitants.
Many economic impacts are difficult to quantify and valuation also may be problematic. Such factors
as animal welfare, human health and the environment are of obvious importance, but do not have
market values and different people have different perceptions of their value. It is therefore impossible
to provide objective assessments of the total cost of most animal diseases, especially the most
serious ones that have wide-ranging effects. But the cost of animal disease can be enormous.